CN116669774A

CN116669774A - Compositions and methods for treating brile disease

Info

Publication number: CN116669774A
Application number: CN202180081451.0A
Authority: CN
Inventors: J·奥尔多
Original assignee: University of Pennsylvania Penn
Current assignee: University of Pennsylvania Penn
Priority date: 2020-10-09
Filing date: 2021-10-08
Publication date: 2023-08-29

Abstract

Provided herein are polynucleotide sequences encoding functional human alpha-galactosidase a (hGLA) and expression cassettes containing these coding sequences. Also provided are vectors, such as recombinant adeno-associated virus (rAAV) vectors having a vector genome comprising an hGLA encoding sequence operably linked to one or more regulatory sequences. In addition, compositions containing these expression cassettes and rAAV, and methods of treating fabry disease using these compositions, are provided.

Description

Compositions and methods for treating brile disease

Background

Fabry disease is an X-linkedLysosomal disorders are defined by the enzyme responsible for acyl sphingosine trihexoses (GL-3 or Gb ₃ ) The gene mutation of the degrading enzyme alpha-galactosidase A (GLA). The deficiency of GLA results in the accumulation of GL-3 and related glycosphingolipids in the plasma and lysosomes of whole blood vessels, nerves, tissues and organs. The disease is a systemic disease and is manifested by progressive renal failure, heart disease, cerebrovascular disease, peripheral nerve disease of small fiber, skin lesions and other abnormalities. Mutation of the GLA gene resulting in a loss of alpha-galactosidase a activity leads to a typical, severe form of fabry disease. Mutations that reduce but small eliminate enzyme activity typically result in milder, delayed fabry disease that typically affects only the heart or kidneys.

Currently, standard treatments for fabry disease include enzyme replacement therapies and drugs to treat and prevent other symptoms of the disease. Kidney transplantation may be required in severe cases where renal failure occurs.

There is a need in the art for compositions and methods for safe and effective treatment of patients with british disease.

Disclosure of Invention

In one aspect, provided herein is a recombinant AAV (rAAV) comprising an AAVhu68 capsid having packaged therein a vector genome, wherein the vector genome comprises a coding sequence for functional human α -galactosidase a (hGLA) and regulatory sequences that direct expression of hGLA in a target cell, wherein the coding sequence comprises SEQ ID NO:4 to 1287, or a sequence at least 85% identical thereto, and wherein hGLA has a cysteine residue at position 233 and/or position 359. In certain embodiments, hGLA comprises at least SEQ ID NO:2, or a sequence at least 95% identical thereto. In certain embodiments, hGLA comprises SEQ ID NO:7 from amino acids 32 to 429. In certain embodiments, wherein hGLA comprises a natural signal peptide. In other embodiments, hGLA comprises a heterologous signal peptide. In certain embodiments, hGLA comprises SEQ ID NO:17 (amino acids 1 to 429), or a sequence at least 95% identical thereto. In certain embodiments, the vector genome comprises a tissue-specific promoter. In certain embodiments, the regulatory sequences comprise a CB7 promoter, an intron, and a polyA. In certain embodiments, the regulatory sequences comprise woodchuck hepatitis virus posttranscriptional regulatory elements (WPREs). In certain embodiments, the vector genome comprises one or more miRNA target sequences.

In one aspect, provided herein is an expression cassette comprising a nucleic acid sequence encoding a functional human α -galactosidase a (hGLA) and one or more regulatory sequences that direct expression of hGLA in a target cell containing the expression cassette, wherein the nucleic acid sequence comprises SEQ ID NO:4 to 1287, or a sequence at least 85% identical thereto, and wherein hGLA has a cysteine residue at position 233 and/or position 359. In certain embodiments, hGLA comprises SEQ ID NO:7 from amino acids 32 to 429. In certain embodiments, hGLA comprises a natural signal peptide. In other embodiments, hGLA comprises a heterologous signal peptide. In certain embodiments, hGLA comprises SEQ ID NO:7 (amino acids 1 to 429), or a sequence at least 95% identical thereto. In certain embodiments, the expression cassette according to any one of claims 12 to 16, wherein the expression cassette comprises a tissue specific promoter. In certain embodiments, the regulatory sequences comprise a CB7 promoter, an intron, and a polyA. In certain embodiments, the regulatory sequences comprise woodchuck hepatitis virus posttranscriptional regulatory elements (WPREs). In certain embodiments, the expression cassette comprises one or more miRNA target sequences.

In one aspect, provided herein is a plasmid comprising an expression cassette comprising a nucleic acid sequence encoding a functional human α -galactosidase a (hGLA) and one or more regulatory sequences that direct expression of hGLA in a target cell containing the expression cassette, wherein the nucleic acid sequence comprises SEQ ID NO:4 to 1287, or a sequence at least 85% identical thereto, and wherein hGLA has a cysteine residue at position 233 and/or position 359. In certain embodiments, the expression cassette is flanked by AAV 5 'itrs and AAV 3' itrs. In a further embodiment, a host cell comprising an expression cassette or plasmid is provided.

In yet another aspect, a pharmaceutical composition comprising a rAAV or an expression cassette comprising a nucleic acid sequence encoding a functional human α -galactosidase a (hGLA) is provided.

In another aspect, a method of treating a human subject diagnosed with GLA deficiency (fabry disease) is provided, comprising administering to the subject a pharmaceutical composition comprising a rAAV or an expression cassette having a sequence encoding functional human α -galactosidase a (hGLA). In another aspect, rAAV, expression cassettes, and pharmaceutical compositions for treating GLA deficiency (fabry disease) are provided.

Other aspects and advantages of the present invention will become apparent from the following detailed description of the invention.

Drawings

FIG. 1 shows a map of CB7.CI. HGLaco (D237C1359C). WPRE.rBG vector genome (SEQ ID NO: 6).

FIG. 2 shows a map of the CB7.CI. HGLANat. WPRE. RBG vector genome (SEQ ID NO: 10).

FIG. 3 shows a map of the TBG.PI.hGLANat.WPRE.bGH vector genome (SEQ ID NO: 8).

FIG. 4 shows a map of the CB7.CI. HGLaco. WPRE. RBG vector genome (SEQ ID NO: 14).

FIG. 5 shows a map of the TBG.PI.hGLaco.WPRE.bGH vector genome (SEQ ID NO: 12).

FIG. 6 shows a map of CB7.CI. HGLaco (M51C_G360C). WPRE.rBG vector genome (SEQ ID NO: 18).

FIG. 7 shows a map of the TBG.PI.hGLaco (M51C_G360℃) WPRE.bGII vector genome (SEQ ID NO: 16).

FIGS. 8A and 8B show the nucleotide sequence alignment of hGLANat (SEQ ID NO: 1), hGLASO (SEQ ID NO: 3), hGLASO (M51C_G360C) (SEQ ID NO: 5), hGLA (D233C_I359C) (SEQ ID NO: 4).

FIG. 9 shows amino acid sequence alignment of hGLASAT (SEQ ID NO: 2), hGLASO (SEQ ID NO: 13), hGLASO (M51C_G360C) (SEQ ID NO: 17), hGLA (D233 C_I 359C) (SEQ ID NO: 7).

FIGS. 10A and 10B show body weights of untreated male and female control, gla KO, WT/TgG S and Gla KO/TgG S mice. Age-matched controls (WT male and Gla HET female), gla KO, WT/TgG3S and Gla KO/TgG S mice were left untreated to evaluate the natural history of these models. The body weights of male (fig. 10A) and female (fig. 10B) mice at 6 weeks of age, 12 weeks of age, 18 weeks of age, 25 weeks of age, 30 weeks of age, and 35 weeks of age were recorded. Average body weight is given. Error bars represent standard deviation. Abbreviations: gla, α -galactosidase a; tgG3S, human Gb3 synthase-transgenic.

FIGS. 11A and 11B show the hotplate response latency of untreated male and female control, gla KO, WT/TgG S and Gla KO/TgG S mice. Age-matched controls (WT male and Gla HET female), glaKO, WT/TgG3S and Gla KO/TgG S mice were left untreated to evaluate the natural history of these models. Sensitivity to thermal stimuli in each mouse model was recorded as response latency (seconds) using a hotplate assay at weeks 6, 12, 18, 25, 30 and 35. Data are presented as average recordings of male (fig. 11A) and female (fig. 11B) mice at various time points. Error bars represent standard error of the mean.

FIGS. 12A and 12B show Blood Urea Nitrogen (BUN) concentrations of untreated male and female controls, gla KO, WT/TgG S and Gla KO/TgG S mice. Age-matched controls (WT male and Gla HET female), gla KO, WT/TgG3S and Gla KO/TgG S mice were left untreated to evaluate the natural history of these models. Blood urea nitrogen concentrations (mg/dL) were recorded at weeks 6, 12, 18, 25, 30 and 35. Data are presented as average recordings of male (fig. 12A) and female (fig. 12B) mice at various time points. Error bars represent standard error of the mean.

Fig. 13A and 13B show urine osmolarity measured in male and female control, gla KO, tgG S and Gla KO/TgG S mice. Age-matched controls (WT male and Gla HET female), gla KO, WT/TgG3S and Gla KO/TgG S mice were left untreated to evaluate the natural history of these models. Urine osmotic pressure (mOsm/kg) was measured at week 25, week 30 and week 35. Data are presented as average recordings of male (fig. 13A) and female (fig. 13B) mice at various time points. Error bars represent standard error of the mean.

FIGS. 14A and 14B show GL-3 storage in kidneys of male Gla KO, WT/TgG3S and Gla KO/TgG 3S. Age-matched controls (WT male and Gla HET female), gla KO, WT/TgG3S, gla KO/TgG S mice were left untreated to evaluate the natural history of these models. Kidneys and hearts were harvested at necropsy and stained with antibodies (dark pellet) and nuclear counterstains that recognize GL-3. (fig. 14A) shows representative IHC images from male mice. Arrows in the kidney images represent the stored material in the glomeruli. The dashed circle areas show lesions of interstitial mononuclear inflammation (nephritis) seen only in some Gla KO/TgG3S mice. Arrows in the cardiac image represent myocardial cell necrosis and mineralization immediately adjacent to myocardial cells with GL-3 storage. FIG. 14B is a bar graph showing the quantification of GL-3 storage (area percent) in whole kidneys using immunohistochemical data. Results for male Gla KO, WT/TgG S and Gla KO/TgG S mice are shown. * P < 0.01, based on the Kruskal-Wallis test (Kruskal-Wallis test) comparing each group with WT/TgG3S control.

FIGS. 15A and 15B show GL-3 storage in the Dorsal Root Ganglion (DRG) of male Gla KO, WT/TgG3S and Gla KO/TgG 3S. Age-matched controls (WT male and Gla HET female), gla KO, WT/TgG3S, gla KO/TgG S mice were left untreated to evaluate the natural history of these models. DRGs were collected at necropsy and stained with antibodies recognizing GL-3 and nuclear counterstains. (fig. 15A) shows a representative image from a male mouse. FIG. 15B is a bar graph showing the quantification of GL-3 storage (area percent) in DRG using immunohistochemical data. Results for male Gla KO, WT/TgG S and Gla KO/TgG S mice are shown. * P < 0.01, based on the krueschel-wales test comparing each group to WT/TgG3S control.

FIGS. 16A to 16D show quantification of lyso-Gb3 in plasma and GL-3 in tissues by LC-MS/MS in Gla KO, tgG S and Gla KO/TgG S mice. Age-matched controls (WT male and Gla HET female), gla KO, WT/TgG3S, gla KO/TgG S mice were left untreated to evaluate the natural history of these models. Kidney, heart and brain tissue and plasma were collected at necropsy. LC-MS/MS was used to quantify GL3 in kidney (FIG. 16A), heart (FIG. 16B) and brain tissue (FIG. 16C) or lyso-Gb3 in plasma (FIG. 16D). For each graph, males and females are plotted separately, with female data at the bottom. * p < 0.05, < p < 0.01 krusec-wales test.

FIG. 17 shows transgene product expression (GLA enzyme activity) measured in Gla/mouse serum on day 7 after administration of control (PBS) or one of the three AAVhu68.HGLA vectors. At 1x10 ¹¹ GC(5.0x10 ¹² GC/kg) or 5X10 ¹¹ GC(2.5x10 ¹³ GC/kg) to male and female mice IV of 2 to 3 months of age were dosed with PBS (control) or AAVhu68.HGLANAT, AAVhu68.HGLACO or AAVhu68.HGLACO (M51C_G360C). Blood was collected at week 1 for serum separation and analyzed for GLA activity. The left panel shows summary data for all animals, and the right and bottom panels show the results divided by gender.

FIG. 18 shows the administration of control (PBS) or one of the three AAVhu68.HGLA vectors followed by Gla ^-/- Biodistribution of AAV genomic DNA measured in mice. At 1x10 ¹¹ GC(5.0x10 ¹² GC/kg) or 5X10 ¹¹ GC(2.5x10 ¹³ GC/kg) to male and female mice IV of 2 to 3 months of age were dosed with PBS (control) or AAVhu68.HGLANAT, AAVhu68.HGLACO or AAVhu68.HGLACO (M51C_G360C). Liver samples were collected at necropsy and analyzed for carrier distribution. Results are expressed as GC of transgene specific sequences relative to the amount of cellular genomic DNA.

FIG. 19 shows transgene product expression (GLA enzyme activity) levels in Gla KO mouse hearts 28 days after administration of one of the three AAVhu68.CB7.HGLA vectors. At 1x10 ¹¹ GC(5.0x10 ¹² GC/kg) or 5X10 ¹¹ GC(2.5x10 ¹³ GC/kg) to male and female mice IV of 2 to 3 months of age were dosed with PBS (control) or AAVhu68.HGLANAT (hGLA), AAVhu68.HGLACO or AAVhu68.HGLACO (M51C_G360C). Cardiac samples were collected at necropsy and analyzed for GLA activity levels. The left panel shows summary data for all animals, and the middle and right panels show the results divided by gender.

FIG. 20 shows transgene product expression (GLA enzyme activity) in liver of Gla KO mice 28 days after administration of one of the three AAVhu68.CB7.hGLA vectors. At 1x10 ¹¹ GC(5.0x10 ¹² GC/kg) or 5X10 ¹¹ GC(2.5x10 ¹³ GC/kg) dosage PBS (control) or AAVhu68.HGLANAT (hGLA), AAVhu68.HGLACO or AAVhu68.HGLACO (M51C_G360C) was administered IV to male and female mice of 2 to 3 months of age). Liver samples were collected at necropsy and analyzed for GLA activity levels. The left panel shows summary data for all animals, and the middle and right panels show the results divided by gender.

FIG. 21 shows transgene product expression (GLA enzyme activity) in the kidney of Gla KO mice 28 days after administration of one of the three AAVhu68.CB7.hGLA vectors. At 1x10 ¹¹ GC(5.0x10 ¹² GC/kg) or 5X10 ¹¹ GC(2.5x10 ¹³ GC/kg) to male and female mice IV of 2 to 3 months of age were dosed with PBS (control) or AAVhu68.HGLANAT (hGLA), AAVhu68.HGLACO or AAVhu68.HGLACO (M51C_G360C). Kidney samples were collected at necropsy and analyzed for GLA activity levels. The left panel shows summary data for all animals, and the middle and right panels show the results divided by gender.

FIG. 22 shows transgene product expression (GLA enzyme activity) in Gla KO mouse brain 28 days after administration of one of the three AAVhu68.CB7.hGLA vectors. At 1x10 ¹¹ GC(5.0x10 ¹² GC/kg) or 5X10 ¹¹ GC(2.5x10 ¹³ GC/kg) to male and female mice IV of 2 to 3 months of age were dosed with PBS (control) or AAVhu68.HGLANAT (hGLA), AAVhu68.HGLACO or AAVhu68.HGLACO (M51C_G360C). Brain samples were collected at necropsy and analyzed for GLA activity levels. The left panel shows summary data for all animals, and the middle and right panels show the results divided by gender.

FIG. 23 shows transgene product expression (GLA enzyme activity) in the small intestine of Gla KO mice 28 days after administration of one of the three AAVhu68.CB7.HGLA vectors. At 1x10 ¹¹ GC(5.0x10 ¹² GC/kg) or 5X10 ¹¹ GC(2.5x10 ¹³ GC/kg) to male and female mice IV of 2 to 3 months of age were dosed with PBS (control) or AAVhu68.HGLANA (hGLA), AAVhu68.HGLACO or AAVhu68.HGLACO (M51C_G360C). Small intestine samples were collected at necropsy and analyzed for GLA activity levels. The upper graph shows summary data for all animals, and the middle and lower graphs show the results divided by gender.

FIG. 24 shows lyso-Gb3 storage (trihexitoyl sphingosine) in Gla KO mouse plasma and GL-3 storage in heart and kidney tissue after administration of one of the three AAVhu68.CB7.HGLA vectors. At 1x10 ¹¹ GC(5.0x10 ¹² GC/kg) or 5X10 ¹¹ GC(2.5x10 ¹³ GC/kg) to male and female mice IV of 2 to 3 months of age were dosed with PBS (control) or AAVhu68.HGLANAT (hGLA), AAVhu68.HGLACO or AAVhu68.HGLACO (M51C_G360C). Plasma was collected and the amount of storage material lyso-Gb3 was measured by LC-MS/MS (upper panel). Kidney and heart samples were collected at necropsy and analyzed for GL-3 storage levels (middle and lower panels, respectively). The upper graph shows summary data for all animals, and the middle and lower graphs show the results divided by gender.

FIG. 25 shows transgene product expression (GLA enzyme activity) in Gla KO mouse serum following administration of AAV vectors or vehicles. At 2.5x10 ¹² GC/kg (Low dose LD), 5.0x10 ¹² GC/kg (medium dose; MD), or 2.5X10 ¹³ GC/kg (high dose; HD), AAVhu68.HGLACO (WTCO), AAVhu68.HGLACO (M51C_G360C) (AT#1) or AAVhu68.HGLACO (D233C_I 359C) (AT#2) was administered to adult (3.5 to 4.5 month old) male and female Gla KO or WT mice IV for only the dose of AAVhu68.HGLACO (D233C_I 359C). Additional Gla KO or WT mice IV administration vehicle (PBS) served as control. Serum samples were collected 7 days after administration and analyzed for transgene product expression (GLA enzyme activity). Summary data for all animals is provided, as is data divided by gender. The results of vehicle-treated WT and Gla KO mice are historical data and are included for reference. Historical Gla enzyme activity values from both WT and Gla KO mouse samples were below quantifiable limits, so no data points were plotted. HD, high dose; LD, low dose; MD, medium dose.

Figure 26 shows transgene product expression (Gla enzyme activity) in Gla KO mouse plasma after administration of AAV vector or vehicle. At 2.5x10 ¹² GC/kg (Low dose; LD), 5.0x10 ¹² GC/kg (medium dose; MD), or 2.5X10 ¹³ GC/kg (high dose; HD), AAVhu68.HGLACO (WTCO), AAVhu68.HGLACO (M51C_G360C) (AT#1) or AAVhu68.HGLACO (D233C_I 359C) (AT#2) was administered to adult (3.5 to 4.5 month old) male and female Gla KO or WT mice IV for only the dose of AAVhu68.HGLACO (D233C_I 359C). Additional Gla KO or WT mice IV administration vehicle (PBS) served as control. Plasma samples were collected 28 days after injection and analyzed for transgene product expression (GLA enzyme activity). The upper diagram shows a stationSummary data for animals, middle and lower panels show the results divided by gender.

FIG. 27 shows transgene product expression (GLA enzyme activity) in the heart of Gla KO mice following administration of AAV vectors or vectors. At 2.5x10 ¹² GC/kg (Low dose; LD), 5.0x10 ¹² GC/kg (medium dose; MD), or 2.5X10 ¹³ GC/kg (high dose; HD), AAVhu68.HGLACO (WTCO), AAVhu68.HGLACO (M51C_G360C) (AT#1) or AAVhu68.HGLACO (D233C_I 359C) (AT#2) was administered to adult (3.5 to 4.5 month old) male and female Gla KO or WT mice IV for only the dose of AAVhu68.HGLACO (D233C_I 359C). Additional Gla KO or WT mice IV administration vehicle (PBS) served as control. Cardiac samples were collected at necropsy and analyzed for transgene product expression (GLA enzyme activity). The upper graph shows summary data for all animals, and the middle and lower graphs show the results divided by gender. The results of vehicle-treated WT and Gla KO mice are historical data and are included for reference. Historical Gla enzyme activity values from both WT and Gla KO mouse samples were below quantifiable limits, so no data points were plotted.

FIG. 28 shows transgene product expression (GLA enzyme activity) in liver of Gla KO mice following administration of AAV vectors or vehicles. At 2.5x10 ¹² GC/kg (Low dose; LD), 5.0x10 ¹² GC/kg (medium dose; MD), or 2.5X10 ¹³ GC/kg (high dose; HD), AAVhu68.HGLACO (WTCO), AAVhu68.HGLACO (M51C_G360C) (AT#1) or AAVhu68.HGLACO (D233C_I 359C) (AT#2) was administered to adult (3.5 to 4.5 month old) male and female Gla KO or WT mice IV for only the dose of AAVhu68.HGLACO (D233C_I 359C). Additional Gla KO or WT mice IV administration vehicle (PBS) served as control. Liver samples were collected at necropsy and analyzed for transgene product expression (GLA enzyme activity). The upper graph shows summary data for all animals, and the middle and lower graphs show the results divided by gender. The results of vehicle-treated WT and Gla KO mice are historical data and are included for reference. Historical Gla enzyme activity values from both WT and Gla KO mouse samples were below quantifiable limits, so no data points were plotted.

FIG. 29 shows transgene product expression (GLA enzyme) in the kidney of Gla KO mice following administration of AAV vectors or vectorsActivity). At 2.5x10 ¹² GC/kg (Low dose; LD), 5.0x10 ¹² GC/kg (medium dose; MD), or 2.5X10 ¹³ GC/kg (high dose; HD), AAVhu68.HGLACO (WTCO), AAVhu68.HGLACO (M51C_G360C) (AT#1) or AAVhu68.HGLACO (D233C_I359C) (AT#2) was administered to adult (3.5 to 4.5 month old) male and female Gla KO or WT mice IV for only the dose of AAVhu68.HGLACO (D233C_1359C). Additional Gla KO or WT mice IV administration vehicle (PBS) served as control. Kidney samples were collected at necropsy and analyzed for transgene product expression (GLA enzyme activity). The upper graph shows summary data for all animals, and the middle and lower graphs show the results divided by gender. The results of vehicle-treated WT and Gla KO mice are historical data and are included for reference. Historical Gla enzyme activity values from both WT and Gla KO mouse samples were below quantifiable limits, so no data points were plotted.

FIG. 30 shows lyso-Gb in plasma collected from Gla KO mice after administration of AAV vectors or vehicles ₃ (trihexyphenyl) storage. At 2.5x10 ¹² GC/kg (Low dose; LD), 5.0x10 ¹² GC/kg (medium dose; MD), or 2.5X10 ¹³ GC/kg (high dose; HD), AAVhu68.HGLACO (WTCO), AAVhu68.HGLACO (M51C_G360C) (AT#1) or AAVhu68.HGLACO (D233C_I 359C) (AT#2) was administered to adult (3.5 to 4.5 month old) male and female Gla KO or WT mice IV for only the dose of AAVhu68.HGLACO (D233C_I 359C). Additional Gla KO or WT mice IV administration vehicle (PBS) served as control. Plasma samples were collected at necropsy 28 days after administration and analyzed for lyso-Gb ₃ Storage level. The upper graph shows summary data for all animals, and the middle and lower graphs show the results divided by gender. The results of vehicle-treated WT and Gla KO mice are historical data and are included for reference.

FIGS. 31A and 31B show GL-3 (acyl sphingosine triose) storage in Gla KO mouse kidneys after administration of AAV vectors or vehicles. At 2.5x10 ¹² GC/kg (Low dose; LD), 5.0x10 ¹² GC/kg (medium dose; MD), or 2.5X10 ¹³ GC/kg (high dose; HD) was administered IV to adult (3.5 to 4.5 month old) male and female Gla KO or WT mice only for doses of AAVhu68.HGLaco (D237C1359C)Aavhu68.Hglaco, aavhu68.Hglaco (m51c_g360C) (eng#1) or aavhu68.Hglaco (d233c_1359c) (eng#2). Additional Gla KO or WT mice IV administration vehicle (PBS) served as control. (FIG. 31A) kidneys were harvested at necropsy and stained with antibodies recognizing GL-3 (acyl sphingosine trihexose, arrow). Representative images from males are shown and labeled. FIG. 31B is a bar graph providing GL-3+ IHC signal quantification, showing the percentage of tubules with GL-3+ deposits. * p < 0.05, p < 0.01, p < 0.001, p < 0.0001 based on the krueschel-wales test followed by a post-mane multiplex comparison test comparing each group to vehicle treated Gla KO mice.

FIGS. 32A and 32B show GL-3 (acyl sphingosine trihexose) storage in the DRG of Gla KO after administration of AAV vectors or vehicles. At 2.5x10 ¹² GC/kg (Low dose; LD), 5.0x10 ¹² GC/kg (medium dose; MD), or 2.5X10 ¹³ GC/kg (high dose; HD), AAVhu68.HGLACO (D233C_I 359 Cco) or AAVhu68.HGLACO (M51C_G360C) (eng#1) or AAVhu68.HGLACO (D233C_I 359C) (eng#2) was administered to adult (3.5 to 4.5 month old) male and female Gla KO or WT mice IV for only the dose of AAVhu68.HGLACO (D233C_I 359 Cco). Additional Gla KO or WT mice IV administration vehicle (PBS) served as control. (FIG. 32A) DRGs were collected at necropsy along with spinal cord and stained with antibodies recognizing GL-3 (acyl sheath saddle triose, dark precipitate). Representative images from males are shown and labeled. FIG. 32B is a bar graph showing the quantized GL-3+ IHC signal in percent GL-3+ area. * p < 0.05, p < 0.01, p < 0.001, p < 0.0001 based on the krueschel-wales test, followed by comparison of each group with the post-prandial dunn multiplex comparison test of vehicle-treated Gla KO mice.

FIG. 33 shows Western blot analysis of GLA secreted in vivo in plasma from AAV-treated animals administered AAV hu68.HGLACO (hGLACO), AAV hu68.HGLACO (M51C_G360C) (hGLACend#1) or AAV hu68.HGLACO (D233C_I 359C) (hGLACend#2).

Fig. 34A and 34B show cardiac transduction and expression of hGLA by immunohistochemical staining in Gla KO male mice (fig. 34A) and female mice (fig. 34B) treated with aavhu68.Hglaco (d233 c_i 359C). By usingLow dose-LD (2.5x10) ¹² GC/kg), mid-dose-MD (5X 10) ¹² 12 GC/kg) or high dose-HD (2.5x10) ¹³ GC/kg), AAVhu68.HGLACO (M51C_G360C) or AAVhu68.HGLACO (D233C_I 359C) IV were injected with GLA KO Fabry disease mice 3.5 to 4.5 months of age. Mice were euthanized 4 weeks after injection and tissues were collected. Hearts were fixed with zinc-formalin and embedded in paraffin. Antibodies to hGLA were used to stain transgene expression. Representative pictures of aavhu68.cb7.hglaco (d233 c_i 359C) injected animals are shown. Dark immunostaining of hGLA showed strong and dose-dependent transgene expression in cardiac myocytes from the ventricles and atria.

Fig. 35 shows IV administration LD (2.5x10 ¹² GC/kg)、MD(5x10 ¹² GC/kg) or HD (2.5x10) ¹³ GC/kg), AAVhu68.HGLACO (hGLACO), AAVhu68.HGLACO (M51C_G360C) (hGLACENG#1) or AAVhu68.HGLACO (D233C_I 359C) (hGLA eng#2).

Fig. 36A and 36B show AST and ALT concentrations in adult NHP after a single IV dose of aavhu68.Hglaco (d233 c_i 359C) (hglaeng#2). Adult NHP (n=4) received a single IV administration of aavhu68.Hglaco (d233 c_i 359C) (hGLA eng#2) at a dose of 2.5x10 ¹³ GC/kg. Blood was collected at baseline, day 0, day 3, day 7, day 14, day 28, and day 60 and analyzed for AST (fig. 36A) and ALT (fig. 36B) concentrations. The dashed line represents the reference value. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; GC, genome copy; GGT, gamma-glutamyl transferase.

Figures 37A-37C show total bilirubin (TBil) levels, platelet counts and White Blood Cell (WBC) counts in adult NHPs after a single IV dose of aavhu68.Hglaco (d233 c_i 359C) (hGLAeng # 2). Adult NHP (n=4) received a single IV administration of aavhu68.Hglaco (d233 c_i 359C) (hglaeng#2) at a dose of 2.5x10 ¹³ GC/kg. Blood was collected at baseline, day 0, day 3, day 7, day 14, day 28, and day 60 and analyzed for TBil levels (fig. 37A), platelet counts (fig. 37B), and WBC counts (fig. 37C). The dashed line represents the reference value.

FIGS. 38A-38C show AAVhu68.HGLACO (D233 C_I 359C) (hGLASENG#2) post-adult NHP with a single Iv dosePT (prothrombin time), APTT (activated partial thromboplastin time) and D-dimer levels. Adult NHP (n=4) received a single IV administration of aavhu68.Hglaco (d233 c_i 359C) (hglaeng#2) at a dose of 2.5x10 ¹³ GC/kg. Blood was collected at baseline, day 0, day 3, day 7, day 14, day 28, and day 60 and analyzed for PT (fig. 38A), APTT (fig. 38B), and D-dimer levels (fig. 38C).

FIG. 39 shows neutralizing and non-neutralizing binding antibodies in adult NHPs after a single IV dose of AAVhu68.HGLaco (D233 C_I 359C) (hGLA eng#2). Abbreviations: babs = non-neutralizing binding antibody; f = female; ID = identity; m = male; nab = neutralizing antibody; NHP = non-human primate. The a-value is the reciprocal serum dilution at 50% reduction of Relative Luminescence Units (RLU) compared to virus control wells (no test sample). The b-value is the reciprocal of the highest serum dilution yielding an average OD450 value 3 times higher than the negative control serum. c-IgG and IgM are BAbs.

FIG. 40 shows transgene product expression (GLA enzyme activity) in plasma of adult NHP after a single intravenous administration of AAVhu68.HGLACO (D233 C_I 359C) (hGLA eng#2). Adult NHP (n=4) received a single IV dose of aavhu68.Hglaco (d233 c_i 359C) (hglaeng#2), at a dose of 2.5x10 ¹³ GC/kg. Plasma was collected on day 7, day 14, day 28 and day 60. Transgenic product expression (GLA enzyme activity) was measured. The dashed line represents the baseline titer.

FIG. 41 shows antibodies against the transgene product (anti-GLA antibodies) in adult NHP plasma after a single intravenous administration of AAVhu68.HGLaco (D233 C_I 359C) (hGLA eng#2). Adult NHP (n=4) received a single IV dose of aavhu68.Hglaco (d233 c_i 359C) (hGLA eng#2) at a dose of 2.5x10 ¹³ GC/kg. Plasma was collected on day 7, day 14, day 28 and day 60. Antibodies against the transgene product (anti-GLA antibodies) were measured. The dashed line represents baseline enzyme activity.

Fig. 42A and 42B show transgene product expression (GLA enzyme activity) of adult NHPs in heart, liver and kidney after single intravenous administration of aavhu68.Hglaco (d233 c_i359C) (hglaeng#2). Adult NHP (n=4) received a single IV dose of aavhu68.Hglaco (d233 c_i 359C) (hGLA eng#2) at a dose of2.5x10 ¹³ GC/kg. On day 60, animals were necropsied and hearts, livers and kidneys were collected to measure transgene product expression (GLA enzyme activity) (fig. 42A). Heart tissue from untreated wild-type NHP of the same species (cynomolgus monkey) was provided by BioIVT as a comparison of baseline GLA enzyme activity (dashed line). The fold increase in GLA enzyme activity was calculated based on the measurements (fig. 42B).

FIG. 43 shows representative images of ISH and GLA expressed IHC from kidney, DRG and heart tissue transgressions of NHP after administration of AAVhu68.HGLaco (D233C-I359C) (hGLA eng#2).

FIG. 44 shows representative images of transgene expression (RNAscope probe) and GLA expressed ISH in cardiac tissue from NHP after administration of AAVhu68.HGLaco (D233C-I359C) (hGLA eng#2).

FIG. 45 shows representative images of transgene expression (RNAscope probe) and GLA expressed ISH in DRG from NHP after administration of AAVhu68.HGLaco (D233C-I359C) (hGLA eng#2).

Detailed Description

Provided herein are compositions useful for treating and/or alleviating the symptoms of fabry disease.

Without wishing to be bound by theory, including regular infusion of recombinant human α -Gal a (rha-Gal a), known as Enzyme Replacement Therapy (ERT), is currently the primary treatment option for fabry patients with non-adaptable mutations, whereas patients with adaptable mutations may benefit from ERT and small chaperones. However, rha-Gal a has low physical stability, short circulation half-life, and variable uptake by tissues associated with different diseases, which may limit the efficacy of ERT and cross-correction dependent gene therapy. The compositions provided herein deliver stable hGLA, which is effective for gene therapy, and provides a larger window for enzymes to remain active in circulation before being absorbed into target tissue.

In certain embodiments, the compositions and methods described herein include nucleic acid sequences, expression cassettes, vectors, recombinant viruses, and other compositions and methods for expressing functional hGLA. In certain embodiments, the compositions and methods described herein include nucleic acid sequences, expression cassettes, vectors, recombinant viruses, host cells, other compositions and methods for producing compositions comprising a nucleic acid sequence encoding a functional hGLA or hGLA polypeptide. In yet another embodiment, the compositions and methods described herein include nucleic acid sequences, expression cassettes, vectors, recombinant viruses, other compositions and methods for delivering a nucleic acid sequence encoding a functional hGLA to a subject for treatment of fabry disease. In one embodiment, the compositions and methods described herein can be used to provide therapeutic levels of hGLA in the peripheral (e.g., blood, liver, kidney) and/or peripheral nervous system of a subject. In certain embodiments, the adeno-associated virus (AAV) vector-based methods described herein provide a new therapeutic option that helps restore the desired function of hGLA and alleviate symptoms associated with hGLA deficiency (fabry disease) by providing for expression of hGLA in a subject in need thereof.

As used herein, the term "therapeutic level" means that the enzyme activity is at least about 5%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, greater than 100%, about 2-fold, about 3-fold, or about 5-fold that of a healthy control. Suitable assays for measuring hGLA enzyme activity are known to those skilled in the art. In some embodiments, such therapeutic levels of hGLA may result in relief of symptoms associated with fabry disease; improvement of disease biomarkers associated with fabry disease (e.g., reduction of Gb3 levels in serum, urine, and/or other biological samples); promoting other treatments of fabry disease (e.g., enzyme replacement or chaperone therapy); preventing neurocognitive decline; reversing certain fabry disease-related symptoms and/or preventing progression of fabry disease-related symptoms; or any combination thereof.

As used herein, "healthy control" refers to a subject or a biological sample therefrom, wherein the subject does not suffer from fabry disease or hGLA deficiency. The healthy control may be from one subject. In another embodiment, the healthy control is a pooled sample from multiple subjects.

As used herein, the term "biological sample" refers to any cell, biological fluid, or tissue. Samples suitable for use in the present application may include, but are not limited to, whole blood, leukocytes, fibroblasts, serum, urine, plasma, saliva, bone marrow, cerebrospinal fluid, amniotic fluid and skin cells. Such samples may be further diluted with saline, buffer or physiologically acceptable diluents. Alternatively, these samples are concentrated by conventional methods.

With respect to the description herein, it is intended that each of the carriers and other compositions described herein be useful in another embodiment. In addition, in another embodiment, each of the compositions described herein that can be used in the methods are themselves also embodiments of the present application.

Unless defined otherwise herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs by reference to the disclosure, which provides one of ordinary skill in the art with a general guidance to many terms used in this application.

As used herein, "disease," "disorder," and "condition" refer to fabry disease and/or hGLA deficiency in a subject.

As used herein, the term "fabry disease-related symptoms" or "symptoms" refers to symptoms found in fabry patients as well as animal models of fabry disease. These symptoms include, but are not limited to, angiokeratomas, acroparesthesias, hypohidrosis/anhidrosis, cornea, lens clouding, heart problems, pain, and reduced renal function. Furthermore, common heart-related signs and symptoms of fabry disease include left ventricular hypertrophy, valvular disease (especially mitral valve prolapse and/or regurgitation), early onset coronary artery disease, angina, myocardial infarction, conduction abnormalities, cardiac arrhythmias, congestive heart failure. Fabry disease is referred to by other names, including alpha-galactosidase a deficiency, anderson-fabry disease, and diffuse somatic keratomas.

As used herein, "patient" or "subject" refers to male or female human, dog, and animal models for clinical studies. In certain embodiments, the subject of these methods and compositions is a human diagnosed with fabry disease. In further embodiments, the human subjects of these methods and compositions are prenatal, neonatal, infant, young child, preschool child, school-age child, adolescent, young or adult.

"comprising" is a term that includes other components or method steps. When "comprising" is used, it is understood that the related embodiments include the use of "consisting of" and "consisting essentially of" the description of the term "consisting of" excludes other components or method steps, and "consisting essentially of" the term excludes any components or method steps that substantially alter the nature of the embodiments or inventions. It should be understood that, while various embodiments in the specification are presented using an "comprising" language, in various instances, related embodiments are also described using a "consisting of or" consisting essentially of language.

References to "one embodiment," "another embodiment," or "an embodiment" in describing the embodiments do not imply that the referenced embodiment is mutually exclusive of another embodiment (e.g., the embodiment described before the referenced embodiment), unless explicitly stated otherwise.

It should be noted that the term "a" or "an" refers to one or more, for example, "expression cassette" is understood to mean one or more expression cassettes. Thus, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein.

As used herein, unless otherwise indicated, the term "about" refers to a variability of ±10% compared to the reference given.

1. Human alpha-galactosidase A (hGLA)

As used herein, the terms "human α -galactosidase a" and "hGLA" are used interchangeably to refer to human α -galactosidase a enzyme. Alternative names for alpha-galactosidase a include galactosidase alpha (agalsidase alfa), alpha-D-galactosidase a, alpha-D-galactosidase galactose hydrolase, alpha-galactosidase a, ceramide methyltrisaccharase, GALA, galactosidase, alpha and melibiase. It should be understood that the greek letter "alpha" and the symbol "alpha" may be used interchangeably throughout the specification. Including native (wild-type) hGLA proteins, particularly variant hGLA proteins expressed from the nucleic acid sequences provided herein or functional fragments thereof, which, when delivered in a composition or by the methods provided herein, restore desired function, alleviate symptoms, ameliorate symptoms associated with a fabry-related biomarker (e.g., serum α -GAL), and/or facilitate other treatments for fabry disease.

"human α -galactosidase a" or "hGLA" can be, for example, a full-length protein (including signal peptide and mature protein), a mature protein, a variant protein described herein, or a functional fragment. As used herein, the term "functional hGLA" refers to an enzyme having the amino acid sequence of a full length native (wild-type) protein (as shown in SEQ ID NO:2 and UniProtKB accession No. P06280-1), variants thereof (including those having specific amino acid substitutions described herein), mutants thereof having conservative amino acid substitutions, fragments thereof, variants and fragments thereof having conservative amino acid substitutions, full length or fragments of any combination of mutants that provide at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90% of the native (wild-type) hGLA biological activity level, or about the same, or greater than 100% thereof.

Human alpha-galactosidase A- (SEQ ID NO: 2)Signal peptide (amino acids 1 to 31)

Natural human GLA coding sequence (SEQ ID NO: 1) (see NCBI reference sequence: NM-000169.)Signal peptide (nucleus) Glycoside acids 1 to 93

Reference is made to SEQ ID NO:2, the signal peptide is present at amino acid positions 1 to 31, and the mature protein comprises amino acids 32 to 429. As used herein, "signal peptide" refers to a short peptide (typically about 16 to 35 amino acids) that is present at the N-terminus of a newly synthesized protein. Signal peptides, and in some cases nucleic acid sequences encoding such peptides, may also be referred to as signal sequences, targeting signals, localization sequences, transit peptides, leader sequences, or leader peptides. As described herein, hGLA may comprise a natural signal peptide (i.e., amino acids 1 to 31 of SEQ ID NO: 2) or alternatively a heterologous signal peptide. In certain embodiments, hGLA is a mature protein (lacking a signal peptide sequence).

In certain embodiments, hGLA comprises a heterologous signal peptide. In certain embodiments, such heterologous signal peptides are preferably of human origin and may include, for example, IL-2 signal peptides. Specific heterologous signal peptides useful in certain embodiments include amino acids 1 to 20 from chymotrypsinogen B2, the signal peptide of human alpha-1-antitrypsin, amino acids 1 to 25 from iduronate-2-sulfatase, and amino acids 1 to 23 from a protease CI inhibitor. See, for example, WO2018046774. Other signals/leader peptides may naturally occur in immunoglobulins (e.g., igG), cytokines (e.g., IL-2, IL12, IL18, etc.), insulin, albumin, β -glucuronidase, alkaline protease, or fibronectin secretion signal peptides, etc. See also, for example,

sigalpeptide.de/index.phpm=listspdb_mammalia. Such chimeric hGLA may have a heterologous leader sequence at the position of the entire 31 amino acid native signal peptide. Optionally, the N-terminal truncation of the hGLA enzyme may lack only a portion of the signal peptide (e.g., a deletion of about 2 to about 25 amino acids, or a value therebetween), the entire signal peptide, or a fragment longer than the signal peptide (e.g., up to 70 amino acids based on the numbering of SEQ ID NO: 2. Optionally, the enzyme may contain a C-terminal truncation of about 5, 10, 15, or 20 amino acids in length.

In certain embodiments, a polypeptide having a sequence similar to SEQ ID NO:2 (amino acids 1 to 429) is at least 95% identical, at least 97% identical or at least 99% identical. In certain embodiments, a nucleotide sequence that hybridizes with SEQ ID NO:2 (amino acids 32 to 429) is at least 95%, at least 97% or at least 99% identical. In certain embodiments, a sequence having at least 95% to at least 99% identity to hGLA of full length (amino acids 1 to 429) or mature protein (amino acids 32 to 429) is characterized by having improved biological effects and better safety profile when tested in an appropriate animal model as compared to reference (i.e., natural) hGLA. In certain embodiments, the hGLA enzyme contains modifications at designated positions of the hGLA amino acid sequence. For example, in certain embodiments, the sequence relative to SEQ ID NO:2, hGLA has a cysteine substitution at position 51 and/or position 360. In certain embodiments, the amino acid sequence relative to SEQ ID NO:2, hGLA has a cysteine substitution at position 233 and/or position 359. Examples of such hGLA polypeptides are provided in SEQ ID NO:7 and 17.

As used herein, "conservative amino acid substitution" or "conservative amino acid substitution" refers to the alteration, substitution, or substitution of an amino acid into a different amino acid having similar biochemical properties (e.g., charge, hydrophobicity, and size), as known to those of skill in the art. See also, e.g., FRENCH et al what is a conservative substitution? (What is a conservative substitution); 170 (4): 1459-1472, each of which is incorporated herein by reference in its entirety.

In one aspect, provided herein are nucleic acid sequences encoding functional hGLA proteins, as well as expression cassettes and vectors comprising the same, for example. In one embodiment, the nucleic acid sequence is SEQ ID NO:1, and a wild-type coding sequence reproduced in 1. In a further embodiment, the nucleic acid sequence hybridizes to SEQ ID NO:1, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% identical to the wild-type hGLA sequence of claim 1, and encodes a functional hGLA.

As used herein, "nucleic acid" refers to polymeric forms of nucleotides, including RNA, mRNA, cDNA, genomic DNA, peptide Nucleic Acid (PNA), and synthetic forms and mixed polymers of the foregoing. Nucleotide refers to a ribonucleotide, a deoxynucleotide, or a modified form of either type of nucleotide (e.g., a peptide nucleic acid oligomer). The term also includes DNA in single-stranded and double-stranded form. Those of skill in the art will appreciate that functional variants of these nucleic acid molecules are described herein. Functional variants are nucleic acid sequences that can be directly translated using the standard genetic code to provide an amino acid sequence that is identical to the amino acid sequence translated from the parent nucleic acid molecule.

In certain embodiments, nucleic acid molecules encoding functional hGLA and other constructs described herein can be used to generate expression cassettes and vector genomes, and can be engineered for expression in yeast cells, insect cells, or mammalian cells (e.g., human cells). Methods are known and have been described previously (e.g. WO 96/09378). A sequence is considered engineered if at least one non-preferred codon is replaced with a more preferred codon compared to the wild-type sequence. In this context, a non-preferred codon is a codon that is used in an organism less frequently than another codon encoding the same amino acid, and a more preferred codon is a codon that is used in an organism more frequently than the non-preferred codon. The codon usage frequency of a particular organism can be found in a codon frequency table, such as in www.kazusa.jp/codon. Preferably more than one non-preferred codon, preferably most or all of the non-preferred codons are replaced by more preferred codons. Preferably, codons most commonly used in organisms are used in the engineered sequence. Preferably substitution of codons generally results in higher expression. Those of skill in the art will also appreciate that many different nucleic acid molecules may encode the same polypeptide due to the degeneracy of the genetic code. It will also be appreciated that the skilled artisan can make nucleotide substitutions using conventional techniques that do not affect the amino acid sequence encoded by the nucleic acid molecule to reflect codon usage of any particular host organism in which the polypeptide is expressed. Thus, unless otherwise indicated, a "nucleic acid sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The nucleic acid sequences may be cloned using conventional molecular biology techniques or generated by DNA synthesis from the head, which may be performed by service companies having business in the field of DNA synthesis and/or molecular cloning, such as GeneArt, gold srey company (GenScript), life technologies company (Life Technologies), eurofins, using conventional procedures.

In certain embodiments, the nucleic acids, expression cassettes, vector genomes described herein comprise hGLA coding sequences as engineered sequences. In certain embodiments, the engineered sequences can be used to improve production, transcription, expression, or safety in a subject. In certain embodiments, the engineered sequences can be used to increase the efficacy of the resulting therapeutic composition or treatment. In further embodiments, the engineered sequences can be used to increase the therapeutic efficacy of the expressed functional hGLA protein, and can also allow for lower doses of therapeutic agents that deliver functional hGLA. In certain embodiments, the engineered hGLA coding sequence is characterized by an increased translation rate as compared to the wild-type hGLA coding sequence.

By "engineered" is meant that the nucleic acid sequences encoding the functional hGLA enzymes described herein are assembled and placed into any suitable genetic element, e.g., naked DNA, phage, transposon, cosmid, episome, etc., that transfers the hGLA sequences carried thereon to a host cell, e.g., for the production of a non-viral delivery system (e.g., RNA-based system, naked DNA, etc.), or for the production of a viral vector in a packaging host cell, and/or for delivery to a host cell in a subject. In certain embodiments, the genetic element is a vector. In one embodiment, the genetic element is a plasmid. Methods for preparing such engineered constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., green and Sambrook, molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), cold spring harbor Press (Cold Spring Harbor Press), new York Cold spring harbor (2012).

In the context of nucleic acid sequences, the terms "percent (%)" identity "," percent sequence identity "or" percent identity "refer to the same residues in two sequences when correspondingly aligned. The length of the sequence identity comparison may exceed the full length of the construct, the full length of the gene coding sequence, or a fragment of at least about 500 to 1000 nucleotides. However, identity between smaller fragments may also be desired, e.g., at least about nine nucleotides, typically at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides.

The percent identity of a protein, polypeptide, about 100 amino acids, about 300 amino acids or peptide fragments thereof, or the amino acid sequence over the entire length of the corresponding nucleic acid sequence coding sequence can be readily determined. Suitable amino acid fragments can be at least about 8 amino acids in length, and can be up to about 50 amino acids in length. In general, when referring to "identity", "homology" or "similarity" between two different sequences, the "identity", "homology" or "similarity" is determined with reference to the "aligned" sequences. "aligned" sequences or "alignment" refers to multiple nucleic acid sequences or protein (amino acid) sequences, typically containing deletions or corrections of additional bases or amino acids as compared to a reference sequence.

Identity can be determined by making alignments of sequences and by using various algorithms and/or computer programs (e.g., BLAST, exPASy; clustal Omega; FASTA; using, for example, the Nidlemann-Wen application algorithm, the Smith-Waterman algorithm) known in the art or commercially available. Alignment was performed using any of a variety of published or commercially available multiple sequence alignment programs. Sequence alignment programs can be used for amino acid sequences, such as the "Clustal Omega", "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME" and "Match-Box" programs. Typically, any of these programs is used at default settings, but one skilled in the art can change these settings as desired. Alternatively, one skilled in the art may utilize another algorithm or computer program that provides at least the level of identity or alignment provided by the cited algorithms and programs. See, e.g., j.d.thomson et al, nucleic acids research (nucleic acids res.), "comprehensive comparison of multiple sequence alignments (A comprehensive comparison of multiple sequence alignments), 27 (13): 2682-2690 (1999).

In certain embodiments, the hGLA coding sequence hybridizes to SEQ ID NO:1 and encodes the wild-type hGLA sequence of SEQ ID NO: 2. 7 or 17. In another embodiment, the hGLA coding sequence comprises a nucleotide sequence identical to SEQ ID NO:1 (nt) 94 to 1287 is less than 80% identical and encodes the nucleotide sequence of SEQ ID NO: 2. 7 or 17 from amino acids 32 to 429.

In certain embodiments, the hGLA encoding sequence shares less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 89%, less than about 88%, less than about 87%, less than about 86%, less than about 85%, less than about 84%, less than about 83%, less than about 82%, less than about 81%, less than about 80%, less than about 79%, less than about 78%, less than about 77%, less than about 76%, less than about 75%, less than about 74%, less than about 73%, less than about 72%, less than about 71%, less than about 70%, less than about 69%, less than about 68%, less than about 67%, less than about 66%, less than about 65%, less than about 64%, less than about 63%, less than about 62%, less than about 61%, or the identity with the wild-type hGLA encoding sequence (SEQ ID NO: 1). In other embodiments, the hGLA encoding sequence shares about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 89%, about 88%, about 87%, about 86%, about 85%, about 84%, about 83%, about 82%, about 81%, about 80%, about 79%, about 78%, about 77%, about 76%, about 75%, about 74%, about 73%, about 72%, about 71%, about 70%, about 69%, about 68%, about 67%, about 66%, about 64%, about 63%, about 62%, about 61% or less identity with the wild-type hGLA encoding sequence (SEQ ID NO: 1). In another embodiment, the hGLA coding sequence hybridizes to SEQ ID NO:3 at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical, and the sequence encodes a functional hGLA. Identity may be relative to the sequence encoding full length hGLA (e.g., nt 1 to nt 1287 of SEQ ID NO:1 or 3) or relative to the sequence encoding mature hGLA (e.g., nt 94 to nt 1287 of SEQ ID NO:1 or 3). In certain embodiments, the hGLA encoding sequence comprises SEQ ID NO:3, or a sequence encoding full length hGLA at least 85%, 90%, 95% or 99% identical thereto. In certain embodiments, the hGLA encoding sequence comprises SEQ ID NO:3, or a sequence encoding a functional hGLA that is at least 85%, 90%, 95% or 99% identical thereto.

In certain embodiments, hGLA having amino substitution at position 233 and/or position 359 is provided, wherein the amino substitution is substituted with respect to SEQ ID NO:2, number of full-length natural hGLA. In certain embodiments, hGLA has a cysteine residue at position 233 and/or position 359. In certain embodiments, hGLA comprises SEQ ID NO:7, or a sequence at least 95% identical thereto having cysteine residues at positions 233 and 359. In other embodiments, hGLA comprises SEQ ID NO:7, or a sequence having cysteine residues at positions 233 and 359 that is at least 95% identical thereto. In certain embodiments, a nucleic acid encoding SEQ ID NO:7, or a sequence having cysteine residues at positions 233 and 359 that is at least 95% identical thereto, wherein the coding sequence shares less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 89%, less than about 88%, less than about 87%, less than about 86%, less than about 85%, less than about 84%, less than about 83%, less than about 82%, less than about 81%, less than about 80%, less than about 79%, less than about 78%, less than about 77%, less than about 76%, less than about 75%, less than about 74%, less than about 73%, less than about 72%, less than about 71%, less than about 70%, less than about 69%, less than about 68%, less than about 67%, less than about 66%, less than about 65%, less than about 64%, less than about 63%, less than about 61%, or less than about 61% identical to the wild-type hGLA coding sequence (SEQ ID NO: 1). In other embodiments, a nucleic acid encoding SEQ ID NO:7, or a sequence having cysteine residues at positions 233 and 359 that is at least 95% identical thereto, wherein the coding sequence shares less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 89%, less than about 88%, less than about 87%, less than about 86%, less than about 85%, less than about 84%, less than about 83%, less than about 82%, less than about 81%, less than about 80%, less than about 79%, less than about 78%, less than about 77%, less than about 76%, less than about 75%, less than about 74%, less than about 73%, less than about 72%, less than about 71%, less than about 70%, less than about 69%, less than about 68%, less than about 67%, less than about 66%, less than about 65%, less than about 62%, less than about 64%, or less than about 61% of the same as the wild type coding sequence of mature hGLA (nt 94-to nt 1287). In certain embodiments, there is provided an hGLA coding sequence comprising SEQ ID NO:4, or a sequence that is at least 85%, 90%, 95% or 99% identical thereto, wherein the encoded functional hGLA has cysteine residues at positions 233 and 359. In certain embodiments, the hGLA coding sequence comprises SEQ ID NO: nt 94 to 1287 of 4. In another embodiment, there is provided an hGLA coding sequence comprising SEQ ID NO:4, or a sequence at least 85%, 90%, 95% or 99% identical thereto, wherein the encoded functional hGLA has cysteine residues at positions 233 and 359. In certain embodiments, the hGLA coding sequence comprises SEQ ID NO:4.

In certain embodiments, hGLA having amino substitution at position 51 and/or position 360 is provided, wherein the amino substitution is substituted at position 51 with respect to SEQ ID NO: 2) Is the number of full-length natural hGLA. In certain embodiments, hGLA has a cysteine residue at position 51 and/or position 360. In certain embodiments, hGLA comprises SEQ ID NO:17, or a sequence at least 95% identical thereto having cysteine residues at positions 51 and 360. In other embodiments, hGLA comprises SEQ ID NO:17, or a sequence having cysteine residues at positions 51 and 360 that is at least 95% identical thereto. In certain embodiments, a nucleic acid encoding SEQ ID NO:17, or a sequence having cysteine residues at positions 51 and 360 that is at least 95% identical thereto, wherein the sequence shares less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 89%, less than about 88%, less than about 87%, less than about 86%, less than about 85%, less than about 84%, less than about 83%, less than about 82%, less than about 81%, less than about 80%, less than about 79%, less than about 78%, less than about 77%, less than about 76%, less than about 75%, less than about 74%, less than about 73%, less than about 72%, less than about 71%, less than about 70%, less than about 69%, less than about 68%, less than about 67%, less than about 66%, less than about 65%, less than about 64%, less than about 63%, less than about 61%, or less than about 61% identical to the wild-type hGLA encoding sequence (SEQ ID NO: 1). In other embodiments, a nucleic acid encoding SEQ ID NO:17, or a sequence having cysteine residues at positions 51 and 360 that is at least 95% identical thereto, wherein the sequence shares less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 89%, less than about 88%, less than about 87%, less than about 86%, less than about 85%, less than about 84%, less than about 83%, less than about 82%, less than about 81%, less than about 80%, less than about 79%, less than about 78%, less than about 77%, less than about 76%, less than about 75%, less than about 74%, less than about 73%, less than about 72%, less than about 71%, less than about 70%, less than about 69%, less than about 68%, less than about 67%, less than about 66%, less than about 65%, less than about 62%, less than about 61%, or less than about 61% identical to the wild type coding sequence of mature hGLA (SEQ ID NO: 1). In certain embodiments, there is provided an hGLA coding sequence comprising SEQ ID NO:5, or a sequence that is at least 85%, 90%, 95% or 99% identical thereto, wherein the encoded functional hGLA has cysteine residues at positions 51 and 360. In certain embodiments, the hGLA coding sequence comprises SEQ ID NO: nt 94 to nt 1287 of 5. In another embodiment, there is provided an hGLA coding sequence comprising SEQ ID NO:5, or a sequence at least 85%, 90%, 95% or 99% identical thereto, wherein the encoded functional hGLA has cysteine residues at positions 51 and 360. In certain embodiments, the hGLA coding sequence comprises SEQ ID NO:5.

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As used herein, "desired function" means that hGLA enzyme activity is at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% of that of a healthy control.

As used herein, the phrases "alleviating symptoms" and "ameliorating symptoms" and grammatical variations thereof refer to reversing, slowing or preventing the progression of, symptoms associated with fabry disease. In certain embodiments, alleviating or ameliorating means that after administration of the composition or using the method, the total number of symptoms in the patient is reduced by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% compared to before administration or use. In another embodiment, alleviating or ameliorating means that the severity or progression of symptoms is reduced by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% after administration of the composition or using the method, as compared to before administration or use.

Other hGLA variants are also suitable. See also international patent application number PCT/US2019/05567 filed on 10 months 2019, 10, the entire contents of which are incorporated herein by reference.

It will be appreciated that the functional hGLA or compositions in the hGLA coding sequence described herein are intended to apply to other compositions, protocols, aspects, embodiments and methods described in the specification.

2. Expression cassette

In certain embodiments, provided herein are expression cassettes having an engineered nucleic acid sequence encoding functional hGLA and regulatory sequences directing expression thereof. In other embodiments, the expression cassette has an engineered nucleic acid sequence described herein encoding a functional hGLA and regulatory sequences directing its expression.

As used herein, the term "expression" or "gene expression" refers to the process by which information from a gene is used to synthesize a functional gene product. The gene product may be a protein, peptide or nucleic acid polymer (e.g., RNA, DNA or PNA).

As used herein, an "expression cassette" refers to a nucleic acid polymer comprising a coding sequence (including variants and fragments thereof) and a promoter of functional hGLA. In other embodiments, the expression cassette includes one or more regulatory sequences in addition to the promoter. In certain embodiments, the expression vector is a vector genome. In certain embodiments, the expression cassette or vector genome is packaged into a vector. In certain embodiments, plasmids comprising the expression cassettes described herein are provided.

As used herein, the term "regulatory sequence" or "expression control sequence" refers to nucleic acid sequences, such as initiation sequences, enhancer sequences, and promoter sequences, which induce, inhibit, or control transcription of a nucleic acid sequence encoding a protein operably linked thereto.

As used herein, the term "operably linked" refers to an expression control sequence adjacent to a nucleic acid sequence encoding hGLA and/or an expression control sequence that functions in trans or at a distance to control transcription and expression thereof.

The term "heterologous" when used in reference to a protein or nucleic acid in a plasmid, expression cassette or vector means that the protein or nucleic acid is present with another sequence or subsequence that are not in the same relationship to each other in nature.

In certain embodiments, provided expression cassettes include a promoter that is a chicken β -actin promoter. Various chicken beta-actin promoters have been described, alone or in combination with various enhancer elements (e.g., CB7 is chicken beta-actin promoter with cytomegalovirus enhancer element, CAG promoter, including promoter, first exon and first intron of chicken beta actin, and splice acceptor of rabbit beta-globin Gene), CBh promoter [ SJ Gray et al, human Gene therapy (Hu Gene thor), 2011, month 9; 22 (9): 1143-1153]. In other embodiments, suitable promoters may include, but are not limited to, the elongation factor 1 alpha (EF 1 alpha) promoter (see, e.g., kimdw et al, gene (Gene) 1990, 7 months 16 using the Human elongation factor 1 alpha promoter as a universal and efficient expression system (Use of the Human elongation factor 1 alpha promoter as a versatile and efficient expression system); 91 (2): 217-23), a synapsin 1 promoter (see, e.g., kugler S et al, human synapsin 1 Gene promoter confers high neuronal specificity for adenovirus vectors according to transduction regions in adult rat brain, long term transgene expression (Human synopsin 1 Gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area), "Gene therapy (Gene Ther.),; 10 (4): 337-47), a Neuronal Specific Enolase (NSE) promoter (see, e.g., kim J et al, cholesterol-rich lipid raft involved in interleukin-6 induced neuroendocrine differentiation of LNCaP prostate cancer cells (Involvement of cholesterol-rich lipid rafts in interleukin-6-induced neuroendocrine differentiation of LNCaP prostate cancer cells), endocrinology (Endocrinology),; 145 (2): 613-9. Electronic version, 10 month 16 days 2003), or a CB6 promoter (see, e.g., large Scale production of serum type 9 adeno-associated Virus vector carrying the human surviving motor neuron Gene (Large-Scale Production of Adeno-Associated Viral Vector Serotype-9Carrying the Human Survival Motor Neuron Gene), molecular biotechnology (Mol Biotechnol.) (2016, 1 month; 58 (1): doi:10.1007/s 12033-015-9899-5).

Examples of tissue specific promoters are well known for liver and other tissues (albumin, miyatakc et al, (1997) journal of virology (J. Virol.), 71:5124-32, hepatitis B virus core promoter, sandig et al, (1996) Gene therapy (3:1002-9), alpha Fetoprotein (AFP), arbuthenot et al, (1996) human Gene therapy (7:1503-14), osteocalcin (Stein et al, (1997) molecular biology report (mol. Biol. Rep.)), 24:185-96); bone sialoprotein (Chen et al, (1996) journal of bone mineral research (J.Bone Miner. Res.), "11:654-64"), lymphocytes (CD 2, hansal et al, (1998) journal of immunology (J.Immunol.), "161:1063-8; immunoglobulin heavy chain; T cell receptor chain), neuronal (e.g., neuron Specific Enolase (NSE)) promoters (Andersen et al, (1993) journal of cell molecular neurobiology (cell. Mol. Neurobiol.)," 13:503-15), neurofilament light chain genes (Piccioli et al, (1991) journal of national academy of sciences (Proc. Natl. Acad. Sci. USA), 88:5611-5) and neuronal specific vgf genes (Piccioli et al, (1995) neuronal (Neuron) 15:373-84), and the like. In certain embodiments, the promoter is a human thyroxine-binding globulin (TBG) promoter. Alternatively, a regulatable promoter may be selected. See, for example, WO 2011/126808B2, which is incorporated herein by reference.

In certain embodiments, the expression cassette comprises one or more expression enhancers. In certain embodiments, the expression cassette contains two or more expression enhancers. These enhancers may be the same or different. For example, enhancers may include the αmic/bik enhancer or the CMV enhancer. Such enhancers may be present in two copies adjacent to each other. Alternatively, the two copies of an enhancer may be separated by one or more sequences. In a further stepIn embodiments, the expression cassette further comprises introns, such as chicken β -actin intron, human β -globulin intron, SV40 intron, and/or commercially available intronsIntrons. Other suitable introns include those known in the art, for example, as described in WO 2011/126808.

The provided expression cassettes may include one or more expression enhancers, such as post-transcriptional regulatory elements from woodchuck hepatitis virus (WPRE), human hepatitis virus (HPRE), ground pine hepatitis virus (GPRE), or arctic pine hepatitis virus (AGSPRE); or synthetic post-transcriptional regulatory elements. These expression enhancing elements are particularly advantageous when placed in the 3' utr and can significantly increase mRNA stability and/or protein yield. In certain embodiments, provided expression cassettes include regulatory sequences that are woodchuck hepatitis virus post-transcriptional regulatory elements (WPREs) or variants thereof. Suitable WPRE sequences are provided in the vector genomes described herein and are known in the art (e.g., as described in U.S. Pat. nos. 6,136,597, 6,287,814 and 7,419,829, which are incorporated herein by reference). In certain embodiments, WPRE is a variant that has been mutated to eliminate expression of the woodchuck hepatitis B virus X (WHX) protein, including, for example, a mutation in the initiation codon of the WHX gene (see, zanta-Boussif et al, gene therapy, month 5 2009; 16 (5): 605-19, incorporated herein by reference). In certain embodiments, the WPRE comprises SEQ ID NO: 27. In other embodiments, the enhancer is selected from non-viral sources.

In addition, the provided expression cassettes include suitable polyadenylation signals. In certain embodiments, the polyA sequence is rabbit β -globin polyA. See, for example, WO 2014/151341. In another embodiment, the polyA sequence is bovine growth hormone polyA. Alternatively, another polyA is included, such as human growth hormone (hGH) polyadenylation sequence, S450 polyA, or synthetic polyA.

In certain embodiments, the expression cassette may include one or more miRNA (also referred to as miR or microrna) target sequences in the untranslated region. The miRNA target sequence is designed to be specifically recognized by a miRNA present in a cell where transgene expression is undesirable and/or where reduced levels of transgene expression are desirable. In certain embodiments, the expression cassette comprises a miRNA target sequence that specifically reduces hGLA expression in the dorsal root ganglion. In certain embodiments, the miRNA target sequence is located in the 3'utr, the 5' utr, and/or both the 3 'and 5' utr of the expression cassette. In certain embodiments, the expression cassette comprises at least two tandem repeats of a Dorsal Root Ganglion (DRG) -specific miRNA target sequence, wherein the at least two tandem repeats comprise at least a first miRNA target sequence and at least a second miRNA target sequence, which may be the same or different. In certain embodiments, the start of the first of the at least two DRG-specific miRNA tandem repeats is within 20 nucleotides of the 3' end of the hGLA encoding sequence. In certain embodiments, the start point of the first of the at least two DRG-specific miRNA tandem repeats is at least 100 nucleotides from the 3' end of the hGLA encoding sequence. In certain embodiments, the miRNA tandem repeat sequence comprises a length of 200 to 1200 nucleotides. In certain embodiments, the inclusion of a miR target does not alter the expression or therapeutic effect of the therapeutic transgene in one or more target tissues relative to an expression cassette lacking the miR target sequence.

In certain embodiments, the expression cassette contains at least one miRNA target sequence which is a miR-183 target sequence. In certain embodiments, the expression cassette contains a miR-183 target sequence that includes AGTGAATTCTACCAGTGCCATA (SEQ ID NO: 31), wherein the sequence complementary to the miR-183 seed sequence is underlined. In certain embodiments, the expression cassette contains more than one copy (e.g., two or three copies) of a sequence 100% complementary to the miR-183 seed sequence. In certain embodiments, the miR-183 target sequence is about 7 nucleotides to about 28 nucleotides in length and comprises at least one region that is at least 100% complementary to a miR-183 seed sequence. In certain embodiments, the miR-183 target sequence comprises a nucleotide sequence that hybridizes to SEQ ID NO:31, and thus, when complementary to SEQ ID NO:31, there are one or more mismatches. In certain embodiments, the miR-183 target sequence comprisesAnd SEQ ID NO:31, wherein the mismatches may be non-contiguous. In certain embodiments, the miR-183 target sequence comprises a region of 100% complementarity, which also comprises at least 30% of the length of the miR-183 target sequence. In certain embodiments, the region of 100% complementarity comprises a sequence having 100% complementarity to a miR-183 seed sequence. In certain embodiments, the remainder of the miR-183 target sequence has at least about 80% to about 99% complementarity to miR-183. In certain embodiments, the expression cassette comprises a miR-183 target sequence comprising a truncated SEQ ID NO:31, i.e. in SEQ ID NO:31 or at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides of sequence deleted at either or both of the 5 'or 3' ends. In certain embodiments, the expression cassette comprises a transgene and one miR-183 target sequence. In other embodiments, the expression cassette comprises at least two, three, or four miR-183 target sequences. In certain embodiments, the inclusion of at least two, three, or four miR-183 target sequences in an expression cassette results in increased levels of transgene expression in a target tissue (e.g., heart).

In certain embodiments, the expression cassette contains at least one miRNA target sequence, which is a miR-182 target sequence. In certain embodiments, the expression cassette contains a miR-182 target sequence, which includes AGTGTGAGTTCTACCATTGCCAAA (SEQ ID NO: 32). In certain embodiments, the expression cassette contains more than one copy (e.g., two or three copies) of a sequence 100% complementary to miR-182 sequences. In certain embodiments, the miR-182 target sequence is about 7 nucleotides to about 28 nucleotides in length and comprises at least one region that is at least 100% complementary to the miR-182 seed sequence. In certain embodiments, the miR-182 target sequence contains a sequence that hybridizes to SEQ ID NO:32, and thus, when complementary to SEQ ID NO:32, there are one or more mismatches. In certain embodiments, the miR-183 target sequence comprises the sequence as set forth in SEQ ID NO:32 sequences having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches when aligned, wherein the mismatches may be non-contiguous. In certain embodiments, the miR-182 target sequence comprises a region of 100% complementarity, which also comprises at least 30% of the length of the miR-182 target sequence. In certain embodiments, the region of 100% complementarity comprises a sequence having 100% complementarity to a miR-182 seed sequence. In certain embodiments, the remainder of the miR-182 target sequence has at least about 80% to about 99% complementarity to miR-182. In certain embodiments, the expression cassette comprises a miR-182 target sequence comprising a truncated SEQ ID NO:32, i.e., in SEQ ID NO:32, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the sequence are deleted at either or both of the 5 'or 3' ends. In certain embodiments, the expression cassette comprises a transgene and one miR-182 target sequence. In other embodiments, the expression cassette comprises at least two, three, or four miR-182 target sequences.

The term "tandem repeat" is used herein to refer to the presence of two or more consecutive miRNA target sequences. These miRNA target sequences may be contiguous, i.e., directly after each other, such that the 3 'end of one is directly upstream of the 5' end of the next, without an intervening sequence, and vice versa. In another embodiment, two or more miRNA target sequences are separated by a short spacer sequence.

As used herein, a "spacer" is any selected nucleic acid sequence located between two or more consecutive miRNA target sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In certain embodiments, the spacer is 1 to 8 nucleotides in length, 2 to 7 nucleotides in length, 3 to 6 nucleotides in length, four nucleotides in length, 4 to 9 nucleotides in length, 3 to 7 nucleotides in length, or more. Suitably, the spacer is a non-coding sequence. In certain embodiments, the spacer may be four (4) nucleotides. In certain embodiments, the spacer is GGAT. In certain embodiments, the spacer is six (6) nucleotides. In certain embodiments, the spacer is CACGTG or GCATGC.

In certain embodiments, the tandem repeat sequence contains two, three, four or more identical miRNA target sequences. In certain embodiments, the tandem repeat sequence contains at least two different miRNA target sequences, at least three different miRNA target sequences, or at least four different miRNA target sequences, and the like. In certain embodiments, the tandem repeat sequence may contain two or three identical miRNA target sequences and a different fourth miRNA target sequence.

In certain embodiments, at least two different sets of tandem repeat sequences may be present in an expression cassette. For example, a 3'UTR may contain a tandem repeat sequence immediately downstream of the transgene, a UTR sequence, and two or more tandem repeat sequences near the 3' end of the UTR. In another example, the 5' utr may contain one, two or more miRNA target sequences. In another example, the 3'utr may contain a tandem repeat sequence and the 5' utr may contain at least one miRNA target sequence.

In certain embodiments, the expression cassette contains two, three, four or more tandem repeats that start within about 0 to 20 nucleotides of the stop codon of the transgene. In other embodiments, the expression cassette contains a miRNA tandem repeat sequence of at least 100 to about 4000 nucleic acids from the transgene stop codon.

See also international patent application numbers PCT/US19/67872 filed on 12 months 20 2019 and international patent application number PCT/US21/32003 filed on 12 months 5 years 2021, the entire contents of which are incorporated herein by reference.

It should be understood that the compositions in the expression cassette are intended to apply to other compositions, protocols, aspects, embodiments, and methods described in the specification.

3. Carrier body

In one aspect, provided herein are vectors comprising a nucleic acid sequence encoding a functional hGLA. In certain embodiments, the vector comprises an expression cassette for delivering an hGLA encoding sequence as described herein.

As used herein, a "vector" is a biological or chemical moiety comprising a nucleic acid sequence that can be introduced into a suitable target cell for replication or expression of the nucleic acid sequence. Examples of vectors include, but are not limited to, recombinant viruses, plasmids, liposomes, multimers, dendrimers, cell-penetrating peptide (CPP) conjugates, magnetic particles, or nanoparticles. In certain embodiments, the vector is a nucleic acid molecule into which an engineered nucleic acid encoding a functional hGLA can be inserted, which can then be introduced into a suitable target cell. These vectors preferably have one or more origins of replication, and one or more sites at which recombinant DNA can be inserted. Vectors generally have means by which cells with the vector can be selected from cells without the vector, e.g., they encode a drug resistance gene. Common vectors include plasmids, viral genomes, and "artificial chromosomes". Conventional methods for generating, producing, characterizing or quantifying the vector are available to those skilled in the art

In certain embodiments, the vector is a non-viral plasmid comprising an expression cassette described herein (e.g., "naked DNA," "naked plasmid DNA," RNA, and mRNA, which can be conjugated to various compositions and nanoparticles, including, for example, micelles, liposomes, cationic lipid-nucleic acid compositions, glycan compositions, and other polymers, lipid-and/or cholesterol-based nucleic acid conjugates), and other constructs as described herein. See, for example, x.su et al, mol. Pharmaceuticals, 2011,8 (3), pages 774 to 787; and (3) network publishing: 21 days of 2011, 3 months; WO2013/182683, WO 2010/053572 and WO 2012/170930, all of which are incorporated herein by reference.

In certain embodiments, the vectors described herein are "replication defective viruses" or "viral vectors," which refer to synthetic or artificial viral particles in which an expression cassette containing a nucleic acid sequence encoding hGLA is packaged in a viral capsid or envelope, wherein any viral genomic sequence that is also packaged within the viral capsid or envelope is replication defective; that is, they are unable to produce progeny virions, but retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not include genes encoding enzymes required for replication (the genome may be engineered to be "cell-free" —only contain nucleic acid sequences encoding hGLA flanked by signals required for amplification and packaging of the artificial genome), but these genes may be provided during production. It is therefore considered safe for use in gene therapy, since replication and infection of progeny virions only occurs in the presence of viral enzymes required for replication.

As used herein, a recombinant viral vector is an adeno-associated virus (AAV), adenovirus, bocavirus, hybrid AAV/bocavirus, herpes simplex virus, or lentivirus.

In certain embodiments, host cells having a nucleic acid comprising an hGLA encoding sequence are provided. In certain embodiments, the host cell contains a plasmid having the hGLA coding sequence as described herein.

As used herein, the term "host cell" may refer to a packaging cell line in which a vector (e.g., recombinant AAV) is produced. The host cell may be a prokaryotic or eukaryotic cell (e.g., human, insect, or yeast) containing exogenous or heterologous DNA that has been introduced into the cell by any means, such as electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion techniques, high-speed DNA coated particles, viral infection, and protoplast fusion. Examples of host cells may include, but are not limited to, isolated cells, cell cultures, E.coli (Escherichia coli) cells, yeast cells, human cells, non-human cells, mammalian cells, non-mammalian cells, insect cells, HEK-293 cells, liver cells, kidney cells, central nervous system cells, neurons, glial cells, or stem cells.

In certain embodiments, the host cell contains an expression cassette for producing hGLA such that the protein is produced in sufficient quantity in vitro for isolation or purification. In certain embodiments, the host cell contains an expression cassette encoding hGLA (including, for example, a functional fragment thereof). As provided herein, hGLA polypeptides may be included in a pharmaceutical composition for administration to a subject as a therapeutic agent (i.e., enzyme replacement therapy).

As used herein, the term "target cell" refers to any cell in which expression of functional hGLA is desired. In certain embodiments, the term "target cell" is intended to refer to a cell of a subject undergoing treatment for fabry disease. Examples of target cells may include, but are not limited to, hepatocytes, kidney cells, smooth muscle cells, and neurons. In certain embodiments, the vector is delivered ex vivo to the target cell. In certain embodiments, the vector is delivered to the target cell in vivo.

It is to be understood that the compositions in the carrier described herein are intended to apply to other compositions, protocols, aspects, embodiments, and methods described in the specification.

4. Recombinant adeno-associated virus (rAAV)

In certain embodiments, provided herein are rAAV comprising an AAV capsid and a vector genome packaged therein. The vector genome comprises an AAV 5 'Inverted Terminal Repeat (ITR), a nucleic acid sequence encoding a functional hGLA described herein, regulatory sequences that direct expression of hGLA in a target cell, and an AAV 3' ITR. In certain embodiments, the vector genome comprises an expression cassette as provided herein flanked by AAV 5 'itrs and AAV 3' itrs. The rAAV is suitable for treating the treatment of the British disease.

As used herein, "rAAV. HGLA" refers to a rAAV having a vector genome comprising a hGLA coding sequence. "rAAVhu68.HGLA" refers to a rAAV having an AAVhu68 capsid and a vector genome comprising an hGLA coding sequence.

As used herein, "vector genome" refers to a nucleic acid sequence packaged within a vector. In one embodiment, the vector genome refers to a nucleic acid sequence packaged within a rAAV capsid to form a rAAV vector. Such nucleic acid sequences comprise AAV Inverted Terminal Repeats (ITRs). In certain embodiments, the ITRs are from an AAV that is different from the one that provides the capsid. In a preferred embodiment, the ITR sequence from AAV2 or a deleted version thereof (Δitr) can be used to facilitate and speed up regulatory approval. However, ITRs from other AAV sources may be selected. When the source of the ITR is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be referred to as a pseudotyped vector. Typically, the AAV vector genome comprises an AAV 5'ITR, regulatory sequences, an hGLA coding sequence, and an AAV 3' ITR. However, other configurations of these elements are also suitable. Shortened versions of the 5' itr, termed AITR, have been described in which the D sequence and terminal resolution sites (trs) are deleted. In certain embodiments, the vector genome comprises a 130 base pair shortened AAV2 ITR in which the external a element is deleted. During vector DNA amplification using the internal a element as a template, the shortened ITR reverts to a wild-type length of 145 base pairs. In other embodiments, full length AAV 5 'and 3' itrs are used. In certain embodiments, the vector genome comprises one or more miRNA target sequences.

In certain embodiments, rAAV is provided having a vector genome comprising an AAV 5'itr, a promoter, an hGLA coding sequence, a poly a sequence, and an AAV3' itr. In certain embodiments, rAAV is provided having a vector genome comprising an AAV 5'itr, a promoter, an intron, an hGLA coding sequence, a poly a sequence, and an AAV3' itr. In certain embodiments, rAAV is provided having a vector genome comprising an AAV 5'itr, a promoter, an hGLA coding sequence, WPRE, a poly a sequence, and an AAV3' itr. In certain embodiments, rAAV is provided having a vector genome comprising an AAV 5'itr, a promoter, an intron, an hGLA coding sequence, WPRE, poly a sequence, and AAV3' itr. In certain embodiments, the vector genome has an enhanced progeny replacing WPRE elements from a non-viral source.

In certain embodiments, rAAV is provided having a vector genome comprising an AAV 5'itr, chicken β -actin intron, hGLA coding sequence, WPRE, poly a sequence, and AAV3' itr. In certain embodiments, rAAV is provided having a vector genome comprising an AAV 5'itr, a CB7 promoter, a chicken β -actin intron, an hGLA encoding sequence, WPRE, a rabbit β -globin poly a sequence, and an AAV3' itr. In certain embodiments, rAAV is provided having a vector genome comprising an AAV 5'itr, a TBG promoter, a chicken β -actin intron, an hGLA encoding sequence, WPRE, a bovine growth hormone poly a sequence, and an AAV3' itr. In certain embodiments, rAAV is provided having a vector genome comprising an AAV 5'ITR, a TBG promoter, SV40 inclusion, hGLA coding sequence, WPRE, bovine growth hormone poly a sequence, and AAV3' ITR. In certain embodiments, the vector genome has an enhanced progeny replacing WPRE element from a non-viral source.

In certain embodiments, rAAV is provided having a vector genome comprising an AAV 5'itr, a promoter, chicken β -actin intron, hGLA encoding sequence, poly a sequence, and AAV 3' itr. In certain embodiments, rAAV is provided having a vector genome comprising an AAV 5'itr, a CB7 promoter, a chicken β -actin intron, an hGLA encoding sequence, a rabbit globin poly a sequence, and an AAV 3' itr. In certain embodiments, rAAV is provided having a vector genome comprising an AAV 5'itr, a TBG promoter, a chicken β -actin intron, an hGLA encoding sequence, a bovine growth hormone poly a sequence, and an AAV 3' itr. In certain embodiments, rAAV is provided having a vector genome comprising an AAV 5'itr, TBG promoter, SV40 intron, hGLA coding sequence, bovine growth hormone polyA sequence, and AAV 3' itr.

In one embodiment, a rAAV is provided having the amino acid sequence of SEQ ID NO: 6. 8, 10, 12, 14, 16 or 18, or a sequence at least 85% identical thereto.

As used herein, the terms "rAAV" and "artificial AAV" are used interchangeably to refer to, but are not limited to, an AAV comprising a capsid protein and a vector genome packaged therein, wherein the vector genome comprises a nucleic acid heterologous to the AAV. In one embodiment, the capsid protein is a non-naturally occurring capsid. Such artificial capsids may be generated by any suitable technique using selected AAV sequences (e.g., fragments of vp1 capsid protein) in combination with heterologous sequences, which may be obtained from different selected AAV, non-contiguous portions of the same AAV, from a non-AAV viral source, or from a non-viral source. The artificial AAV may be, but is not limited to, a pseudotyped AAV, a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid. Pseudotyped vectors are useful in the present invention in which the capsid of one AAV is replaced with a heterologous capsid protein. In one embodiment, AAV2/5 and AAV2/8 are exemplary pseudotyped vectors. The genetic element of choice may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high speed DNA coated particles, viral infection, and protoplast fusion. Methods for preparing such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., green and Sambrook, molecular cloning: laboratory Manual, cold spring harbor Press, new York Cold spring harbor (2012).

The term "AAV" as used herein refers to naturally occurring adeno-associated viruses, adeno-associated viruses obtainable by one of skill in the art and/or obtained according to the compositions and methods described herein, as well as artificial AAV. Adeno-associated virus (AAV) viral vectors are anti-AAV DNase particles having an AAV protein capsid, wherein an expression cassette flanked by AAV Inverted Terminal Repeats (ITRs) is packaged for delivery to a target cell. AAV capsids consist of 60 capsid (cap) protein subunits VP1, VP2 and VP3, which are symmetrically arranged in icosahedron, in a ratio of about 1:1:10 to 1:1:20, depending on the AAV chosen. Various AAV can be selected as a capsid source for the AAV viral vectors identified above. See, for example, U.S. published patent application No. 2007-0036760-A1; U.S. published patent application No. 2009-0197338-A1; EP 1310571. See also, WO 2003/042397 (AAV 7 and other simian AAVs), U.S. Pat. No. 7790449 and U.S. Pat. No. 7282199 (AAV 8), WO 2005/033321 and U.S. Pat. No. 7,906,111 (AAV 9), and WO 2006/110689, and WO 2003/042397 (rh.10). These documents also describe other AAV that may be selected for AAV production and are incorporated herein by reference. Among the AAV isolated or engineered and well characterized from human or non-human primate (NHP), human AAV2 is the first AAV developed as a gene transfer vector; it has been widely used for efficient gene transfer experiments in different target tissues and animal models. Unless otherwise indicated, the AAV capsids, ITRs, and other selected AAV components described herein can be readily selected from any AAV, including, but not limited to, AAV generally identified as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8, and AAVAnc80, AAVhu68, as well as any known or mentioned AAV or yet to be discovered variant or mixture thereof. AAV9 capsids include rAAV with a capsid protein comprising an amino acid sequence that is 9926499% identical to AAS. See also, US7906111 and WO 2005/033321. rAAV with AVVhu68 capsid is described, for example, in WO 2018/160582, which is incorporated herein by reference. In certain embodiments, the capsid protein is designated by a number or combination of numbers and letters following the term "AAV" in the rAAV vector name. See also PCT/US19/19804 and PCT/US19/19861, filed on month 2, 27, 2019, each entitled "novel adeno-Associated Virus (AAV) Vectors, AAV Vectors with reduced capsid deamidation, and uses thereof (novelando-Associated viruses (AAV) Vectors, AAV Vectors Having Reduced Capsid Deamidation And Uses Therefor), which are incorporated herein by reference in their entirety.

As used herein, the term "variant" refers to any AAV sequence derived from a known AAV sequence, including those having conservative amino acid substitutions, and those sharing at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or greater sequence identity with an amino acid or nucleic acid sequence. In another embodiment, an AAV capsid comprises a variant, which may comprise up to about 10% variation from any of the described or known AAV capsid sequences. That is, the AAV capsids share about 90% to about 99.9% identity, about 95% to about 99% identity, or about 97% to about 98% identity with AAV capsids provided herein and/or known in the art. In one embodiment, the AAV capsid shares at least 95% identity with the AAV capsid. When determining the percent identity of AAV capsids, any variable protein (e.g., vp1, vp2, or vp 3) can be compared. "AAV9 variants" as used herein include those described in, for example, WO2016/049230, US 8,927,514, US 2015/0344911 and US 8,734,809.

In certain embodiments, the AAV capsid is selected from the group consisting of natural and engineered clade F adeno-associated viruses. In certain embodiments, a rAAV provided herein comprises an AAVhu68 capsid. AAVhu68 differs from another clade F virus AAV9 within clade F.AAVhu68 (SEQ ID NO: 21) in two encoded amino acids at position 67 and position 157 of vp 1. In contrast, the other clades FAAV (AAV 9, hu31, hu 32) had Ala at position 67 and Ala at position 157. However, in other embodiments, the AAV capsid is selected from a different clade, such as clade A, B, C, D or E, or from an AAV source outside of any of these clades.

rAAVhu68 includes AAVhu68 capsid and vector genome. In one embodiment, the composition comprising rAAVhu68 comprises a combination of a heterologous population of vp1, a heterologous population of vp2, and a heterologous population of vp3 protein. As used herein, the term "heterologous" or any grammatical variant thereof, when used in reference to a vp capsid protein, refers to a population of distinct elements, such as vp1, vp2, or vp3 monomers (proteins) having different modified amino acid sequences. SEQ ID NO:21 provides the coding amino acid sequence of the AAVhu68 vp1 protein. The AAVhu68 capsid contains a subset of the vp1, vp2, and vp3 proteins, which have the sequences from SEQ ID NO:21, and a predicted amino acid residue. These subgroups include at least some deamidated asparagine (N or Asn) residues. For example, certain subgroups are set forth in SEQ ID NO:21 comprises at least one, two, three or four highly deamidated asparagine (N) positions and optionally further comprises other deamidated amino acids, wherein deamidation results in amino acid changes and other optional modifications. Various combinations of these and other modifications are described herein.

As used herein, unless otherwise indicated, a "subpopulation" of vp proteins refers to a group of vp proteins having at least one commonly defined characteristic and consisting of at least one group member to at least all members of a reference group.

For example, unless otherwise indicated, a "subpopulation" of vp1 proteins is at least one (1) vp1 protein and less than all vp1 proteins in the assembled AAV capsid. Unless otherwise indicated, a "subpopulation" of vp3 proteins may be one (1) vp3 protein to at least all vp3 proteins in the assembled AAV capsid. For example, the vp1 protein may be a subset of vp proteins; the vp2 protein may be a separate vp protein sub-population, while vp3 is another vp protein sub-population in the assembled AAV capsid. In another example, vp1, vp2, and vp3 proteins may contain subgroups with different modifications, such as at least one, two, three, or four highly deamidated asparagine, e.g., at an asparagine-glycine pair.

Unless otherwise specified, highly deamidated means at least 45% deamidated, at least 50% deamidated, at least 60% deamidated, at least 65% deamidated, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, up to about 100% deamidated compared to the predicted amino acid sequence at the reference amino acid position (e.g. at least 80% of the asparagine at amino acid 57 of SEQ ID NO:21 may be deamidated based on total vp1 protein, or 20% of the asparagine at amino acid 409 of SEQ ID NO:21 may be deamidated based on total vp1, vp2 and vp3 proteins). Such percentages may be determined using 2D-gel, mass spectrometry techniques, or other suitable techniques.

As provided herein, SEQ ID NO:21 may independently be aspartic acid (Asp), isoaspartic acid (isoAsp), aspartic acid, and/or a tautomeric blend of Asp and isoAsp, or a combination thereof. Any suitable ratio of alpha-and iso-aspartic acids may be present. For example, in certain embodiments, the ratio may be 10:1 to 1:10 aspartic acid: isoaspartic acid, aspartic acid about 50:50: iso-aspartic acid, or about 1:3 aspartic acid: iso-aspartic acid, or another selected ratio. In certain embodiments, SEQ ID NO:21 is deamidated to glutamic acid (Glu), i.e. alpha-glutamic acid, gamma-glutamic acid (Glu), or a blend of alpha-and beta-glutamic acid, which can be interconverted by common glutamine intermediates. Any suitable ratio of alpha-and gamma-glutamic acid may be present. For example, in certain embodiments, the ratio may be from 10:1 to 1:10 α:γ, about 50:50 α:γ, or about 1:3 α:γ, or another selected ratio.

Thus, rAAVhu68 comprises a subpopulation of vp1, vp2, and/or vp3 proteins having deamidated amino acids within the rAAVhu68 capsid, including at least one subpopulation comprising at least one highly deamidated asparagine. In addition, other modifications may include isomerization, particularly at selected aspartic acid (D or Asp) residue positions. In other embodiments, the modification may comprise amidation at the Asp position.

In certain embodiments, the AAVhu68 capsid contains a subpopulation of vp1, vp2, and vp3 having at least 4 to at least about 25 deamidated amino acid residue positions, and SEQ ID NO:21, wherein at least 1 to 10% is deamidated compared to the coding amino acid sequence of 21. Most of these may be N residues. However, the Q residue may also be deamidated.

In certain embodiments, the AAVhu68 capsid is further characterized by one or more of the following. An AAVhu68 capsid protein comprising: by encoding the sequence of SEQ ID NO:21, and the AAVhu68 vp1 protein produced by nucleic acid sequence expression of the predicted amino acid sequences of 1 to 736, from SEQ ID NO:20, or a vp1 protein produced by a sequence that hybridizes to SEQ ID NO:20, which is at least 70% identical, produces a nucleic acid sequence encoding SEQ ID NO:23 to 736, and a vp1 protein of predicted amino acid sequence of 1 to 736; by encoding the sequence of SEQ ID NO:21, and an AAVhu68 vp2 protein produced by expression of a nucleic acid sequence comprising a predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:20 or a vp2 protein produced by a sequence that is identical to at least nucleotides 412 to 2211 of SEQ ID NO:20, and at least nucleotides 412 to 2211 are at least 70% identical, resulting in a nucleic acid sequence encoding SEQ ID NO:21, and/or by encoding a polypeptide from vp2 of predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:21, and an AAVhu68 vp3 protein produced by expression of a nucleic acid sequence comprising the predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:20 or a vp3 protein produced by a sequence that is identical to at least nucleotide 607 to 2211 of SEQ ID NO:20, and at least nucleotides 607 to 2211 are at least 70% identical, resulting in a nucleic acid sequence encoding SEQ ID NO:21 of at least about amino acids 203 to 736.

Additionally or alternatively, there is provided an AAV capsid comprising a heterologous population of vp1 proteins optionally comprising valine at position 157, a heterologous population of vp2 proteins optionally comprising valine at position 157, and a heterologous population of vp3 proteins, wherein the amino acid sequences based on SEQ ID NO:21, at least a subset of the vp1 and vp2 proteins comprise valine at position 157 and optionally further comprise glutamic acid at position 67. Additionally or alternatively, there is provided an AAVhu68 capsid comprising: a heterologous population of vp1 proteins, which encodes SEQ ID NO:21, and a nucleic acid sequence of the amino acid sequence of seq id no; a heterologous population of vp2 proteins, which encodes SEQ ID NO:21 of at least about amino acids 138 to 736; and a heterologous population of vp3 proteins, which is a polypeptide encoding SEQ ID NO:21, wherein at least amino acids 203 to 736, wherein: the vp1, vp2 and vp3 proteins contain subgroups with amino acid modifications.

The AAVhu68 vp1, vp2 and vp3 proteins are typically expressed as encoded by the sequences encoding SEQ ID NO:21 (amino acids 1 to 736). Optionally, the vp1 coding sequence is used alone to express vp1, vp2 and vp3 proteins. Alternatively, the sequence may be identical to the sequence encoding SEQ ID NO:21 (about aa 203 to 736) without the nucleic acid sequence of the vp1 unique region (about aa 1 to about aa 137) and/or the vp2 unique region (about aa 1 to about aa 202), or the strand complementary thereto, the corresponding mRNA (about nt 607 to about nt 2211 of SEQ ID NO: 20) or the sequence complementary thereto: 20 at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO:21 aa 203 to 736. Additionally or alternatively, the vp1 coding and/or vp2 coding sequence may be identical to the sequence encoding SEQ ID NO:21 (about aa 138 to 736) without the nucleic acid sequence of the vp1 unique region (about aa 1 to about 137) or the strand complementary thereto, the corresponding mRNA (nt 412 to 2211 of SEQ ID NO: 20) or the sequence complementary thereto: 20 at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO:21 about aa 138 to 736.

rAAVhu68 has a sequence derived from the expression of the sequence encoding SEQ ID NO:21, and optionally other nucleic acid sequences, e.g., nucleic acid sequences encoding vp3 proteins that do not contain vp1 and/or vp2 unique regions. rAAV hu68 produced using a single nucleic acid sequence vp1 produces a heterologous population of vp1, vp2, and vp3 proteins. More specifically, rAAVhu68 capsids contain a subset of the vp1, vp2, and vp3 proteins, which have the sequences from SEQ ID NOs: 21, and a predicted amino acid residue. These subgroups include at least deamidated asparagine (N or Asn) residues. For example, asparagine in an asparagine-glycine pair is highly deamidated.

In one embodiment, the AAVhu68 vp1 nucleic acid sequence has the sequence of SEQ ID NO:20, or a strand complementary thereto, e.g., the corresponding mRNA. In certain embodiments, the vp2 and/or vp3 proteins may additionally or alternatively be expressed from a different nucleic acid sequence than vp1, e.g., to alter the ratio of vp proteins in a selected expression system. In certain embodiments, there is also provided a nucleic acid encoding SEQ ID NO:21 (about aa 203 to 736) without the nucleic acid sequence of the unique region of vp1 (about aa 1 to about aa 137) and/or the unique region of vp2 (about aa 1 to about aa 202) or the strand complementary thereto, the corresponding mRNA (about nt 607 to about nt 2211 of SEQ ID NO: 20). In certain embodiments, there is also provided a nucleic acid encoding SEQ ID NO:21 (aa 138 to 736) without the nucleic acid sequence of the vp1 unique region (aa 1 to 137) or the complementary strand thereof, the corresponding mRNA (nt 412 to 2211 of SEQ ID NO: 20).

However, the coding sequence of SEQ ID NO:21 are used to generate rAAVhu68 capsids. In certain embodiments, the nucleic acid sequence has the sequence of SEQ ID NO:20, or a nucleic acid sequence that hybridizes to SEQ ID NO:20 at least 70% to 99% identical, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identical to the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the nucleic acid sequence has the sequence of SEQ ID NO:20, or a nucleic acid sequence that hybridizes to SEQ ID NO: about nt 412 to about nt 2211 of 20 is at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence encoding SEQ ID NO:21 (about aa 138 to 736). In certain embodiments, the nucleic acid sequence has the sequence of SEQ ID NO:20 from about nt 607 to about nt 2211, or a nucleic acid sequence identical to SEQ ID NO:20 from about nt 607 to about nt 2211 at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence encoding SEQ ID NO:21 (about aa 203 to 736).

Designing nucleic acid sequences encoding the rAAVhu68 capsid, including DNA (genomic or cDNA) or RNA (e.g., mRNA), is well known in the art Within the domain technology. In certain embodiments, the nucleic acid sequence encoding the AAVhu68 vp1 capsid protein is provided in SEQ ID NO:20. in other embodiments, a sequence corresponding to SEQ ID NO:20 to express AAVhu68 capsid protein. In certain other embodiments, the nucleic acid sequence hybridizes to SEQ ID NO:20 at least about 75% identical, at least 80% identical, at least 85%, at least 90%, at least 95%, at least 97% identical, or at least 99% to 99.9% identical. Such nucleic acid sequences may be codon optimized and may be designed by various methods. For expression in selected systems (i.e., cell types) that can be designed by a variety of methods. Such optimization may be performed using online available methods (e.g., geneArt), published methods, or companies that provide codon optimization services (e.g., DNA2.0 (minipaque, california)). One codon optimization method is described, for example, in U.S. International patent publication No. WO 2015/012924, which is incorporated herein by reference in its entirety. See also, for example, U.S. patent publication No. 2014/0032186 and U.S. patent publication No. 2006/0136814. Suitably, the overall length of the Open Reading Frame (ORF) for the product is modified. However, in some embodiments, only a fragment of the ORF is altered. By using one of these methods, the frequency can be applied to any given polypeptide sequence and produce nucleic acid fragments encoding codon-optimized coding regions for the polypeptide. Many options are available for making actual changes in codons or for synthesizing codon optimized coding regions designed as described herein. Such modifications or syntheses may be performed using standard and conventional molecular biological procedures well known to those of ordinary skill in the art. In one method, a series of complementary oligonucleotide pairs, each 80 to 90 nucleotides in length and spanning the length of the desired sequence, are synthesized by standard methods. These oligonucleotide pairs are synthesized such that upon annealing they form a double-stranded fragment of 80 to 90 base pairs containing cohesive ends, e.g., each oligonucleotide in the pair is synthesized to extend 3, 4, 5, 6, 7, 8, 9, 10 or more bases beyond the region complementary to the other oligonucleotides in the pair. The single-stranded end of each pair of oligonucleotides is designed to anneal to the single-stranded end of the other pair of oligonucleotides. Allowing oligonucleotides Acid pair annealing, then allowing about five to six of these double stranded fragments to anneal together through cohesive single stranded ends, then ligating them together and cloning into a standard bacterial cloning vector, such as that available from Enjetty corporation of Callicarpa, calif. (Invitrogen Corporation)A carrier. The construct is then sequenced by standard methods. Several of these constructs consisting of 80 to 90 base pair fragments, i.e., about 500 base pair fragments, linked together were prepared so that the entire desired sequence was represented in a series of plasmid constructs. The inserts of these plasmids are then cleaved with the appropriate restriction enzymes and ligated together to form the final construct. The final construct was then cloned into a standard bacterial cloning vector and sequenced. Other methods will be apparent to those skilled in the art. In addition, gene synthesis is readily available commercially.

In certain embodiments, asparagine (N) in the N-G pair in the rAAHU 68 vp1, vp2, and vp3 proteins is highly deamidated. In the case of rAAVhu68 capsid protein, 4 residues (N57, N329, N452, N512) generally show deamidation levels > 70% in different batches, and in most cases > 90%. Other asparagine residues (N94, N253, N270, N304, N409, N477 and Q599) also show deamidation levels up to about 20% in different batches. Deamidation levels were initially identified using trypsin digestion and verified with chymotrypsin digestion.

In certain embodiments, the rAAVhu68 capsid contains a subpopulation of AAV vp1, vp2, and/or vp3 capsid proteins having at least four highly deamidated asparagine (N) positions in the rAAVhu68 capsid protein. In certain embodiments, about 20% to 50% of the N-N pairs (excluding the N-N-N triplets) exhibit deamidation. In certain embodiments, the first N is deamidated. In certain embodiments, the second N is deamidated. In certain embodiments, the deamidation is between about 15% and about 25% deamidation. In SEQ ID NO: deamidation at Q at position 259 of 21 is about 8% to about 42% of the AAVhu68 vp1, vp2 and vp3 capsid proteins of the AAVhu68 protein.

In certain embodiments, rAAVhu68 capsid is further characterized by amidation of vp1, vp2, and vp3 proteins in D297. In certain embodiments, the sequence based on SEQ ID NO:21, and about 70% to about 75% of the D at position 297 of the vp1, vp2 and/or vp3 proteins in the AAVhu68 capsid are amidated. In certain embodiments, at least one Asp of vp1, vp2 and/or vp3 of the capsid is isomerized to D-Asp. Based on SEQ ID NO:21, such isomers are typically present in an amount of less than about 1% of Asp at one or more of residue positions 97, 107, 384.

In certain embodiments, rAAVhu68 has an AAVhu68 capsid having vp1, vp2, and vp3 proteins with subgroups comprising combinations of one, two, three, four, or more deamidating residues at the positions listed in the table below. Deamidation in rAAV can be determined using 2D gel electrophoresis and/or mass spectrometry and/or protein modeling techniques. On-line chromatography can be performed with an Acclaim PepMap column and a Thermo UltiMate 3000RSLC system (moeimerfeishi technologies (Thermo Fisher Scientific)) coupled with Q exact HF with a NanoFlex source (zemerfeishi technologies). MS data were obtained using the data dependent top-20 method of Q exact HF, dynamically selecting the most abundant as yet unsequenced precursor ions from the survey scan (200 to 2000 m/z). Sequencing by higher energy collision dissociation fragmentation, target values for 1e5 ions were determined using predictive automatic gain control, and precursor separation was performed using a 4m/z window. The survey scan is obtained at a resolution of 120,000 at m/z 200. The resolution of the HCD spectrum can be set at 30,000 at m/z200, with a maximum ion implantation time of 50ms and a normalized collision energy of 30. The RF level of the S lens was set to 50 to give the best transmission of the m/z region occupied by peptide from digesta. Precursor ions having single, unspecified or six and higher charge states may be excluded from fragmentation selection. BioPharma Finder 1.0 software (Sesamer Feishul technologies) can be used to analyze the data obtained. For peptide mapping, searches were performed using a single order protein FASTA database, in which carbamoyl Methylation is set as a fixed modification; and oxidation, deamidation and phosphorylation were set to variable modifications, mass accuracy of 10-ppm, high protease specificity, and confidence level of MS/MS spectrum of 0.8. Examples of suitable proteases may include, for example, trypsin or chymotrypsin. Mass spectrometry identification of deamidated peptides is relatively simple, since deamidation increases the mass of the intact molecule by +0.984Da (-OH and-NH) ₂ Poor mass between groups). The percentage of deamidation of a particular peptide is determined by dividing the mass area of the deamidated peptide by the sum of the areas of the deamidated peptide and the native peptide. Isobaric species deamidated at different sites can co-migrate in a single peak, taking into account the number of possible deamidation sites. Thus, fragment ions derived from peptides having multiple potential deamidation sites can be used to locate or distinguish between multiple deamidation sites. In these cases, the relative intensities within the observed isotopic patterns can be used to specifically determine the relative abundance of the different deamidated peptide isomers. This method assumes that the fragmentation efficiencies of all isomeric species are the same and independent of deamidation sites. Those skilled in the art will appreciate that many variations of these exemplary methods may be used. Suitable mass spectrometers may include, for example, quadrupole time-of-flight mass spectrometers (QTOF), such as Waters Xevo or Agilent 6530, or Orbitrap instruments, such as Orbitrap Fusion or Orbitrap Velos (thermofisher). Suitable liquid chromatography systems include, for example, the Acquity UPLC system from Waters or Agilent systems (series 1100 or 1200). Suitable data analysis software may include, for example, massLynx (wotery), pinpoint and Pepfinder (zemerter feishi technologies), mascot (Matrix Science), peaks DB (bioinformatics solutions company (Bioinformatics Solutions)). Other techniques may also be described, such as those published on-line at 16 months 6 in 2017, on X.jin et al, methods of human Gene therapy (Hu Gene Therapy Methods), volume 28, phase 5, pages 255 to 267.

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In certain embodiments, the AAVhu68 capsid is characterized by having a capsid protein wherein the amino acid sequence based on SEQ ID NO:21, at least 45% of the N residues are deamidated at least one of positions N57, N329, N452 and/or N512. In certain embodiments, at least about 60%, at least about 70%, at least about 80%, or at least 90% of the N residues at one or more of these N-G positions (i.e., N57, N329, N452 and/or N512, numbering based on the amino acid sequence of SEQ ID NO: 21) are deamidated. In these and other embodiments, the AAVhu68 capsid is further characterized by having a population of proteins wherein about 1% to about 20% of the N residues have deamidation at one or more of the following positions: n94, N253, N270, N304, N409, N477 and/or Q599, based on SEQ ID NO: 21. In certain embodiments, AAVhu68 comprises at least one subpopulation of vp1, vp2, and/or vp3 proteins that are identified in SEQ ID NO:21, N35, N57, N66, N94, N113, N252, N253, Q259, N270, N303, N304, N305, N319, N328, N329, N336, N409, N410, N452, N477, N515, N598, Q599, N628, N651, N663, N709, N735, or a combination thereof. In certain embodiments, the capsid protein may have one or more amidated amino acids.

Other modifications were also observed, most of which did not result in conversion of one amino acid to a different amino acid residue. Optionally, at least one Lys of vp1, vp2 and vp3 of the capsid is acetylated. Optionally, at least one Asp of vp1, vp2 and/or vp3 of the capsid is isomerised to D-Asp. Optionally, at least one S (Ser, serine) of vp1, vp2 and/or vp3 of the capsid is phosphorylated. Optionally, at least one T (Thr, threonine) of vp1, vp2 and/or vp3 of the capsid is phosphorylated. Optionally, at least one W (trp, tryptophan) of vp1, vp2 and/or vp3 of the capsid is oxidized. Optionally, at least one M (Met, methionine) of vp1, vp2 and/or vp3 of the capsid is oxidized. In certain embodiments, the capsid protein has one or more phosphorylations. For example, certain vp1 capsid proteins may be phosphorylated at position 149.

In certain embodiments, the rAAVhu68 capsid comprises a heterologous population of vp1 proteins, the vp1 proteins being encoding SEQ ID NO:21, wherein the vp1 protein comprises glutamic acid (Glu) at position 67 and/or valine (Val) at position 157; a heterologous population of vp2 proteins optionally comprising valine (Val) at position 157; and a heterologous population of vp3 proteins. The AAVhu68 capsid contains at least one subgroup, wherein the sequence based on SEQ ID NO:21, at least 65% of the asparagine (N) in the asparagine-glycine pair at position 57 of the vp1 protein and at least 70% of the asparagine (N) in the asparagine-glycine pair at positions 329, 452 and/or 512 of the vp1, v2 and vp3 proteins are deamidated, wherein deamidation results in an amino acid change. As discussed in more detail herein, deamidated asparagine may be deamidated as aspartic acid, isoaspartic acid, interconverted aspartic acid/isoaspartic acid pairs or combinations thereof. In certain embodiments, rAAVhu68 is further characterized by one or more of: (a) Each of the vp2 proteins is independently at least a polypeptide encoding SEQ ID NO:21, a nucleic acid sequence of the vp2 protein; (b) Each of the vp3 proteins is independently at least a polypeptide encoding SEQ ID NO:21, a nucleic acid sequence of the vp3 protein; (c) the nucleic acid sequence encoding the vp1 protein is SEQ ID NO:21, or a sequence corresponding to SEQ ID NO:20 at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO:21, and a sequence of the amino acid sequence of seq id no. Optionally, the sequences are used alone to express vp1, vp2 and vp3 proteins. Alternatively, the sequence may be identical to the sequence encoding SEQ ID NO:21 (about aa 203 to 736) without the nucleic acid sequence of the vp1 unique region (about aa 1 to about aa 137) and/or the vp2 unique region (about aa 1 to about aa 202), or the strand complementary thereto, the corresponding mRNA (about nt 607 to about nt 2211 of SEQ ID NO: 20) or the sequence complementary thereto: 20 at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO:21 aa 203 to 736. Additionally or alternatively, the vp1 coding and/or vp2 coding sequence may be identical to the sequence encoding SEQ ID NO:21 (about aa 138 to 736) without the nucleic acid sequence of the vp1 unique region (about aa 1 to about 137) or the strand complementary thereto, the corresponding mRNA (nt 412 to 2211 of SEQ ID NO: 20) or the sequence complementary thereto: 20 at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ id NO:21 about aa 138 to 736.

Additionally or alternatively, rAAVhu68 capsid comprises at least a subset of vp1, vp2, and/or vp3 proteins, which are represented in the base SEQ ID NO:21, N57, N66, N94, N113, N252, N253, Q259, N270, N303, N304, N305, N319, N328, N329, N336, N409, N410, N452, N477, N512, N515, N598, Q599, N628, N651, N663, N709, or a combination thereof; (e) The rAAVhu68 capsid comprises a subset of vp1, vp2, and/or vp3 proteins comprising the amino acid sequence based on SEQ ID NO:21 from 1% to 20% deamidation at one or more of positions N66, N94, N113, N252, N253, Q259, N270, N303, N304, N305, N319, N328, N336, N409, N410, N477, N515, N598, Q599, N628, N651, N663, N709, or a combination thereof; (f) The rAAVhu68 capsid comprises a subpopulation of vp1, wherein the nucleic acid sequence based on SEQ ID NO:21, 65% to 100% of the N at position 57 of the vpl protein is deamidated; (g) The rAAVhu68 capsid comprises a subpopulation of vp1 proteins, wherein 75% to 100% of N at position 57 of the vp1 protein is deamidated; (h) The rAAVhu68 capsid comprises a subpopulation of vp1, vp2 and/or vp3 proteins, wherein the nucleic acid sequence based on SEQ ID NO:21, 80% to 100% of N at position 329 is deamidated; (i) The rAAVhu68 capsid comprises a subpopulation of vp1, vp2 and/or vp3 proteins, wherein the nucleic acid sequence based on SEQ ID NO:21, 80% to 100% of the N at position 452 is deamidated; (j) The rAAVhu68 capsid comprises a subpopulation of vp1, vp2 and/or vp3 proteins, wherein the nucleic acid sequence based on SEQ ID NO:21, 80% to 100% of the N at position 512 is deamidated; (k) The rAAV comprises about 60 total capsid proteins in a ratio of about 1vp1 protein: about 1 to 1.5vp2 protein: 3 to 10vp3 protein; (1) rAAV comprises about 60 total capsid proteins, in a ratio of about lvp1 proteins: about 1vp2 protein: 3 to 9vp3 protein.

In certain embodiments, AAVhu68 is modified to alter glycine in the asparagine-glycine pair to reduce deamidation. In other embodiments, asparagine is changed to a different amino acid, such as glutamine deamidated at a slower rate; or amino acids lacking an amide group (e.g., glutamine and asparagine contain an amide group); and/or amino acids lacking an amine group (e.g., lysine, arginine, and histidine contain an amide group). As used herein, amino acids lacking amide or amine side groups refer to, for example, glycine, alanine, valine, leucine, isoleucine, serine, threonine, cystine, phenylalanine, tyrosine or tryptophan, and/or proline. The modification as described may be one, two or three asparagine-glycine pairs found in the encoded AAVhu68 amino acid sequence. In certain embodiments, all four asparagine-glycine pairs are not subjected to such modifications. Thus, a method for reducing deamidation of rAAVhu68 and/or engineered rAAVhu68 variants having lower deamidation rates is provided. In addition, one or more other amide amino acids may be changed to non-amide amino acids to reduce deamidation of rAAVhu 68.

These amino acid modifications can be made by conventional genetic engineering techniques. For example, a nucleic acid sequence comprising a modified AAVhu68 vp codon may be generated, wherein the sequence encoding SEQ ID NO:21 One to three codons of glycine at positions 58, 330, 453 and/or 513 (of the asparagine-glycine pair) are modified to encode amino acids other than glycine. In certain embodiments, the nucleic acid sequence comprising the modified asparagine codon may be found in SEQ ID NO:21, one to three of the asparagine-glycine pairs at positions 57, 329, 452 and/or 512 are engineered such that the modified codon encodes an amino acid other than asparagine. Each modified codon may encode a different amino acid. Alternatively, one or more altered codons may encode the same amino acid. In certain embodiments, these modified AAVhu68 nucleic acid sequences can be used to generate mutant rAAVhu68, whose capsids have a lower deamidation than the native hu68 capsids. Such mutant rAAVhu68 may have reduced immunogenicity and/or increased storage stability, particularly in suspension form. As used herein, "codon" refers to three nucleotides in a sequence encoding an amino acid.

As used herein, an "encoded amino acid sequence" refers to an amino acid predicted based on translation of a known DNA codon of a reference nucleic acid sequence translated into the amino acid. The following table illustrates DNA codons and 20 common amino acids, showing Single Letter Codes (SLC) and three letter codes (3 LC).

As used herein, the term "clade" when referring to an AAV group refers to a set of AAV phylogenetically related to each other as determined by bootstrap values of at least 75% (at least 1000 replicates) and poisson correction distance measurements of no more than 0.05 using a contiguous algorithm based on alignment of AAV vp1 amino acid sequences. The adjacency algorithm has been described in the literature. See, e.g., M.Nei and S.Kumar, molecular evolution and phylogenetic (Molecular Evolution and Phylogenetics) (Oxford university mountain edition (Oxford UniversityPress), new York (2000) such algorithms can be implemented using computer programs, e.g., the MEGAv2.1 program implements the modified Nei-Gojobori method using these techniques and computer programs, and the sequence of the AAVvp1 capsid protein, one of skill in the art can readily determine whether a selected AAV is contained in one of the clades identified herein, in the other clade, or outside of these clades, see, e.g., G Gao et al, J Virol, journal 2004 for 6 months, 78 (10:6381-6388) identifying clades A, B, C, D, E and F, gene bank accession numbers AY530553 through AY530629 see also WO 2005/033321.

Methods of producing capsids and coding sequences thereof, as well as methods of producing rAAV viral vectors, have been described. See, for example, gao et al, proc. Natl. Acad. Sci. USA 100 (10), 6081-6086 (2003) and US 2013/0045186A1.

ITRs or other AAV components can be readily isolated or engineered from AAV using techniques available to those skilled in the art. Such AAV may be isolated, engineered or obtained from an academic, commercial or public source (e.g., american type culture collection, manassas, VA, american Type Culture Collection). Alternatively, AAV sequences may be engineered by synthesis or other suitable methods by reference to published sequences, such as those available in the literature or in databases (e.g., gene banks (GenBank), pubMed, etc.). AAV viruses can be engineered by conventional molecular biology techniques, such that these particles can be optimized for cell-specific delivery of nucleic acid sequences, for minimal immunogenicity, for regulatory stability and particle lifetime, for efficient degradation, for precise delivery to the nucleus, and the like.

In certain embodiments, the rAAV is a self-complementary AAV. "self-complementary AAV" refers to a construct wherein the coding region carried by the recombinant AAV nucleic acid sequence has been designed to form an intramolecular double-stranded DNA template. At the time of infection, the two complementary halves of scAAV will combine to form a double stranded DNA (dsDNA) unit ready for immediate replication and transcription, rather than waiting for cell-mediated synthesis of the second strand. See, e.g., D M McCarty et al, self-complementing recombinant adeno-associated virus (scaV) vectors promote efficient transduction independent of DNA synthesis (Self-complementary recombinant adeno-associated virus (scaV) vectors promote efficient transduction independently of DNA synthesis), gene therapy (month 8 2001), volume 8, 16, pages 1248-1254. Self-complementary AAV is described, for example, in U.S. patent No. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated by reference herein in its entirety.

In certain embodiments, the rAAV is nuclease resistant. Such nucleases can be single nucleases or mixtures of nucleases and can be endonucleases or exonucleases. The nuclease resistant rAAV indicates that AAV capsids have been fully assembled and protects these packaged genomic sequences from degradation (digestion) during a nuclease incubation step designed to remove contaminating nucleic acids that may be present in the production process. In many cases, the rAAV described herein is DNase resistant.

Recombinant adeno-associated viruses (AAV) described herein may be generated using known techniques. See, for example, WO 2003/042397; WO 2005/033321, WO 2006/110689; US 7588772 B2. Such methods involve culturing a host cell containing a nucleic acid sequence encoding an AAV capsid; a functional rep gene; an expression cassette as described herein flanked by AAV Inverted Terminal Repeats (ITRs); and sufficient helper functions to allow the expression cassette to be packaged into an AAV capsid protein. Also provided herein are host cells comprising a nucleic acid sequence encoding an AAV capsid; a functional rep gene; the vector genome; and sufficient helper functions to allow packaging of the vector genome into AAV capsid proteins. In one embodiment, the host cell is a HEK 293 cell. These methods are described in more detail in WO2017160360 A2, which is incorporated herein by reference.

Other methods of producing rAAV available to those skilled in the art may be used. Suitable methods may include, but are not limited to, baculovirus expression systems or production by yeast. See, e.g., robert m.kotin, large-scale recombinant adeno-associated virus production (Large-scale recombinant adeno-associated virus production), human molecular genetics (Hum Mol genet.) 2011, 4 months, 15 days; 20 (R1): R2-R6. On-line publication was made on 2011, 4 and 29. doi:10.1093/hmg/ddr141; aucin MG et al, production of adeno-associated viral vectors in insect cells using triple infection: optimization of baculovirus concentration ratio (Production of adeno-associated viral vectors in insect cells using triple infection: optimization of baculovirus concentration ratios), "biotechnology and bioengineering (Biotechnol bioeng.)," 12/20/2006; 95 (6): 1081-92; SAMI s.thakur, production of recombinant adeno-associated viral vectors in yeast (Production of Recombinant Adeno-associated viral vectors in yeast) submitted to university of florida research institute paper, 2012; kondraov O et al, "Direct Head-to-Head evaluation of recombinant adeno-associated viral vectors prepared in human and insect cells (Direct Head-to-Head Evaluation of Recombinant Adeno-associated Viral Vectors Manufactured in Human versus Insect Cells)," molecular therapy "2017, month 8, and day 10. pii: s1525-0016 (17) 30362-3.doi:10.1016/j. Ymthe.2017.08.003 ] [ electronic plate before print publishing ]; mietzsch M et al, oneBac 2.0: sf9cell line for production of AAV1, AAV2 and AAV8 vectors with minimal exogenous DNA coating (Sf 9Cell Lines for Production of AAV, AAV2, and AAV8 Vectors with Minimal Encapsidation of Foreign DNA), "method of human gene therapy (Hum Gene TherMethods)," month 2 2017; 28 (1): doi:10.1089/hgtb.2016.164; production and characterization of the replication genome of novel recombinant adeno-associated viruses by Li L et al: eukaryotic DNA sources for gene transfer (Production and characterization ofnovel recombinant adeno-associated virus replicative-form genome: a eukaryotic source of DNA for gene transfer), "American science public library (PLoS one.)," 2013, 8 months, 1 day; 8 (8): e69879.doi: 10.1371/journ.fine.0069879.2013 copy; galibert et al, recent progress in large scale production of adeno-associated viral vectors in insect cells for the treatment of neuromuscular diseases (Latest developments in the large-scale production of adeno-associated virus vectors in insect cells toward the treatment of neuromuscular diseases), "journal of invertebrate pathology (J Invertebr pathol.)," 7 th 2011; 107 complement: s80-93.doi:10.1016/j.jip.2011.05.008; and Kotin RM, large-scale recombinant adeno-associated virus production (Large-scale recombinant adeno-associated virus production), human molecular genetics (Hum Mol genet.) "2011, 4 months, 15 days; 20 (R1): r2-6.Doi:10.1093/hmg/ddr141. Electronic version 2011, 4 months and 29 days.

Various methods of AAV purification are known in the art. See examplesWO 2017/160360 entitled "scalable purification method for AAV9 (Scalable Purification Method for AAV)," incorporated herein by reference, describes methods commonly used for clade F capsids. Purification using two-step affinity chromatography followed by anion exchange resin chromatography purification of the carrier drug product and removal of empty capsids. The crude cell collection may undergo steps such as concentration of the carrier collection, diafiltration of the carrier collection, microfluidization of the carrier collection, nuclease digestion of the carrier collection, filtration of the microfluidized intermediates, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and/or formulation and filtration to prepare a batch of carriers. Affinity chromatography purification is performed after anion exchange resin chromatography for purification of the carrier drug product and removal of empty capsids. In one example, for the affinity chromatography step, the diafiltration product may be applied to Capture Select that is effective in capturing AAV2/9 serotypes ^TM Poros-AAV2/9 affinity resin (life technologies Co., ltd. (Life Technologies)). Under these ionic conditions, a significant percentage of residual cellular DNA and protein flows through the column, while AAV particles are effectively captured. See also, WO2021/158915; WO2019/241535; and WO 2021/165537.

Conventional methods of characterizing or quantifying rAAV are available to those skilled in the art. To calculate empty and full particle content, VP3 band volumes of selected samples (e.g., iodixanol gradient purified formulations in the examples herein, where number of GC = number of particles) were plotted against the attached GC particles. The resulting linear equation (y=mx+c) is used to calculate the number of particles in the band volume of the sample peak. The particle count (pt) per 20 μl of loading was then multiplied by 50 to give particles (pt)/mL. Pt/mL divided by GC/mL gives the particle to genome copy ratio (Pt/GC). Pt/mL-GC/mL gave empty Pt/mL. Empty pt/mL divided by pt/mL, x100 gives the percentage of empty particles. In general, methods for assaying empty capsids and AAV vector particles using packaged genomes are known in the art. See, e.g., grimm et al, (1999) 6:1322-1330; sommer et al, (2003) 7:122-128. To test for denatured capsids, the method includesThe treated AAV stock is subjected to SDS-polyacrylamide gel electrophoresis consisting of any gel capable of separating three capsid proteins, e.g. a gradient gel containing 3 to 8% tris-acetate in buffer, then running the gel until the sample material is separated, and blotting the gel onto a nylon or nitrocellulose membrane, preferably nylon. The anti-AAV capsid antibody is then used as a first antibody, preferably an anti-AAV capsid monoclonal antibody, most preferably a B1 anti-AAV-2 monoclonal antibody, that binds to a denatured capsid protein (Wobus et al, (2000) 74:9281-9293). A second antibody is then used which binds to the first antibody and contains means for detecting binding to the first antibody, more preferably an anti-IgG antibody containing a detection molecule covalently bound thereto, most preferably a goat anti-mouse IgG antibody covalently linked to horseradish peroxidase. The method for detecting binding is used for semi-quantitative determination of binding between the first antibody and the second antibody, preferably a detection method capable of detecting radioisotope emissions, electromagnetic radiation or colorimetric changes, most preferably a chemiluminescent detection kit. For example, for SDS-PAGE, samples can be taken from the column fractions and heated in SDS-PAGE loading buffer containing a reducing agent (e.g., DTT), and the capsid proteins resolved on a pre-gradient polyacrylamide gel (e.g., novex). Silver staining may be performed using SilverXpress (Invitrogen), california, according to manufacturer's instructions or other suitable staining methods (i.e. SYPRO ruby or coomassie staining). In one embodiment, the concentration of AAV vector genome (vg) in the column fraction can be measured by quantitative real-time PCR (Q-PCR). The sample is diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA. After nuclease inactivation, taqMan specific for the DNA sequence between the primers and the pair is used ^TM The fluorescent probe further dilutes and amplifies the sample. The number of cycles (threshold cycles, ct) required for each sample to reach a specified fluorescence level was measured on a Applied Biosystems Prism 7700 sequence detection system. A standard curve was generated in a Q-PCR reaction using plasmid DNA containing the same sequence as that contained in the AAV vector. The cycle threshold (Ct) value obtained from the sample was used to normalize it to a plasmidThe Ct values of the standard curve were used to determine vector genome titres. Endpoint analysis based on digital PCR may also be used. As used herein, the terms Genome Copy (GC) and vector genome (vg) are interchangeable in the context of a dose or dose (e.g., GC/kg and vg/kg).

In one aspect, an optimized q-PCR method is used that utilizes a broad spectrum of serine proteases, such as proteinase K (e.g., available from Qiagen). More specifically, the optimized qPCR genome titer assay is similar to the standard assay except that after DNase I digestion, the samples are diluted with proteinase K buffer and treated with proteinase K, then heat-inactivated. Suitably, the sample is diluted with proteinase K buffer in an amount equal to the sample size. Proteinase K buffer may be concentrated to 2-fold or more. Typically, proteinase K treatment is about 0.2mg/mL, but may vary from 0.1mg/mL to about 1 mg/mL. The treatment step is typically conducted at about 55 ℃ for about 15 minutes, but may be conducted at a lower temperature (e.g., about 37 ℃ to about 50 ℃) for a longer period of time (e.g., about 20 minutes to about 30 minutes), or at a higher temperature (e.g., up to about 60 ℃) for a shorter period of time (e.g., about 5 to 10 minutes). Similarly, heat inactivation typically lasts about 15 minutes at about 95 ℃, but may be reduced in temperature (e.g., about 70 to about 90 ℃) and extended in time (e.g., about 20 minutes to about 30 minutes). The sample is then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described in the standard assay.

In addition or alternatively, droplet digital PCR (ddPCR) may be used. For example, methods have been described for determining single stranded and self-complementing AAV vector genome titers by ddPCR. See, e.g., m.lock et al, methods of human gene therapy, month 4 of 2014; 25 (2): doi: 10.1089/hgb.2013.131. Electronic version 2014, 2 month 14 day.

Methods for determining the ratio between vp1, vp2 and vp3 of capsid proteins are also useful. See, e.g., vamseedhar Rayaprolu et al, comparative analysis of adeno-associated viral capsid stability and kinetics (Comparative Analysis of Adeno-Associated Virus Capsid Stability and Dynamics), journal of virology, 2013, month 12; 87 (24): 13150-13160; buller RM, rose JA.1978 characterization of adenovirus-associated virus-induced polypeptides in KB cells (Characterization of adenovirus-associated viruses-induced polypeptides in KB cells), journal of virology 25:331-338; and Rose JA, maizel JV, inman JK, shatkin AJ.1971, structural proteins of adeno-associated viruses (Structural proteins of adenovirus-associated viruses), J.Virol.8: 766-770.

As used herein, the term "treatment" or "treatment" refers to a composition and/or method for the purpose of alleviating one or more symptoms of a solution bri disease, restoring the desired function of hGLA, or ameliorating a biomarker of a disease. In some embodiments, the term "treatment" or "treatment" is defined to encompass administration of one or more compositions described herein to a subject for the purposes indicated herein. Thus, "treating" may include one or more of reducing the onset or progression of fabry disease, preventing disease, reducing the severity of disease symptoms, slowing the progression thereof, eliminating disease symptoms, slowing the progression of disease, or increasing the efficacy of treatment in a given subject.

It should be understood that the compositions in the rAAV described herein are intended to apply to other compositions, protocols, aspects, embodiments, and methods described in the specification.

5. Pharmaceutical compositions or formulations

In certain embodiments, provided herein is a pharmaceutical composition comprising a vector described herein, such as a rAAV, in a formulation buffer. In certain embodiments, the pharmaceutical composition is suitable for co-administration with a functional hGLA protein (ERT) (e.g., fabrzyme) or chaperone therapy (e.g., galafold (migalastat)), aimer (Amicus Therapeutics)). In one embodiment, a pharmaceutical composition is provided that comprises a rAAV as described herein in a formulation buffer. In certain embodiments, the rAAV is at about 1x10 ⁹ Genomic Copy (GC)/mL to about 1x10 ¹⁴ GC/mL formulation. In another embodiment, the rAAV is present at about 3x10 ⁹ GC/mL to about 3x10 ¹³ GC/mL formulation. In yet another embodiment, the rAAV is present at about 1x10 ⁹ GC/mL to about 1x10 ¹³ GC/mL formulation. In one embodiment, the rAAV is at least about 1x10 ¹¹ GC/mL formulation.

In certain embodiments, the pharmaceutical composition comprises an expression cassette comprising the hGLA encoding sequence in a non-viral or viral vector system. This may include, for example, naked DNA, naked RNA, inorganic particles, lipid or lipid-like particles, chitosan-based formulations, and other formulations known in the art and described, for example, by Ramamoorth and Narvekar, as described above). Such non-viral vector systems may include, for example, plasmids or non-viral genetic elements, or protein-based vectors.

In certain embodiments, the pharmaceutical composition comprises a non-replicating viral vector. Suitable viral vectors may include any suitable delivery vector, such as a recombinant adenovirus, a recombinant lentivirus, a recombinant bocavirus, a recombinant adeno-associated virus (AAV), or another recombinant parvovirus. In certain embodiments, the viral vector is a recombinant AAV for delivering hGLA to a patient in need thereof.

As used herein, a rAAV "stock" refers to a rAAV population. Although their capsid proteins are heterogeneous due to deamidation, rAAV in stock are expected to share the same vector genome. The stock may include rAAV with capsids having, for example, heterogeneous deamidation pattern characteristics of the AAV capsid protein of choice and the production system of choice. The stock may be produced by a single production system or pooled by multiple runs of the production system. Various production systems may be selected, including but not limited to those described herein.

In one embodiment, the pharmaceutical composition comprises a vector comprising an expression cassette comprising an hGLA encoding sequence and a formulation buffer suitable for delivery by Intraventricular (ICV), intrathecal (IT), intracisternal, or Intravenous (IV) injection. In one embodiment, the expression cassette comprising the hGLA coding sequence is packaged in a recombinant AAV.

In one embodiment, the pharmaceutical composition comprises a functional hGLA polypeptide or functional fragment thereof for delivery to a subject as Enzyme Replacement Therapy (ERT). Such pharmaceutical compositions are typically administered intravenously, however, in some cases intradermal, intramuscular or oral administration is also possible. The composition can be used for the prophylactic treatment of individuals suffering from or at risk of fabry disease. For therapeutic use, the pharmaceutical composition is administered to a patient suffering from a defined disease in an amount sufficient to reduce the concentration of accumulated metabolites and/or prevent or inhibit further accumulation of metabolites. For individuals at risk of lysosomal enzyme deficiency, the pharmaceutical composition is administered prophylactically in an amount sufficient to prevent or inhibit metabolite accumulation. A pharmaceutical composition comprising the hGLA protein described herein is administered in a therapeutically effective amount. In general, a therapeutically effective amount can vary depending on the severity of the medical condition of the subject as well as the age, general condition, and sex of the subject. The dosage may be determined by a physician and may be adjusted as necessary to suit the observed therapeutic effect. In one aspect, provided herein are pharmaceutical compositions for ERT formulated to contain a unit dose of hGLA protein or functional fragment thereof.

In certain embodiments, the formulation further comprises a surfactant, preservative, excipient, and/or buffer dissolved in the aqueous suspension. In one embodiment, the buffer is PBS. In another embodiment, the buffer is artificial cerebrospinal fluid (aCSF), such as an eioitt formulation buffer (Eliott's formulation buffer); or Harvard instrument perfusion fluid (Harvard apparatus perfusion fluid) (artificial CSF with final ion concentration (in mM): na 150;K 3.0;Ca 1.4;Mg 0.8;P 1.0;C1155). Various suitable solutions are known, including those that include one or more of the following: buffered saline, surfactant and physiologically compatible salt or mixture of salts adjusted to an ionic strength equivalent to about 100mM sodium chloride (NaCl) to about 250mM sodium chloride, or to an ionic concentration equivalent to physiologically compatible salt.

Suitably, the formulation is adjusted to a physiologically acceptable pH, for example in the range of pH 6 to 8, or pH 6.5 to 7.5, pH 7.0 to 7.7, or pH 7.2 to 7.8. Since the pH of cerebrospinal fluid is about 7.28 to about 7.32, for intrathecal delivery, a pH within this range may be desirable; while for intravenous delivery a pH of 6.8 to about 7.2 may be desirable. However, other pH's within the broadest range and these subranges may be selected for other delivery routes.

Suitable surfactants or combinations of surfactants may be selected from non-toxic nonionic surfactants. In one embodiment, a difunctional block copolymer surfactant terminating in primary hydroxyl groups is selected, e.gF68[BASF]Also known as Poloxamer (Poloxamer) 188, which has a neutral pH, has an average molecular weight of 8400. Other surfactants and other poloxamers, i.e., nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly (propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly (ethylene oxide)), SOLUTOL HS 15 (polyethylene glycol-15 hydroxystearate), LABRASOL (polyoxyglyceryl octoate), polyoxyethylene 10 oleyl ether, TWEEN (polyoxyethylene sorbitol fatty acid ester), ethanol, and polyethylene glycol, may be selected. In one embodiment, the formulation contains a poloxamer. These copolymers are generally designated by the letter "P" (for poloxamers) followed by three numbers: the first two numbers x 100 give the approximate molecular weight of the polyoxypropylene core and the last number x 10 gives the percentage of polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005% to about 0.001% of the suspension.

In one example, the formulation may contain, for example, a buffered saline solution comprising one or more of sodium chloride, sodium bicarbonate, dextrose, magnesium sulfate (e.g., magnesium sulfate 7H 2O), potassium chloride, calcium chloride (e.g., calcium chloride 2H 2O), disodium hydrogen phosphate, and mixtures thereof in water. Suitably, for intrathecal delivery, the osmotic pressure is in a range compatible with cerebrospinal fluid (e.g., about 275 to about 290); see, e.g., emedicine. Mediatape. Com/artecle/2093316-overview. Optionally, for intrathecal delivery, a commercially available diluent may be used as a suspending agent, or in combination with another suspending agent and other optional excipients. See, e.g., elliotsSolution [ Lukare medical Co., ltd]。

In certain embodiments, the formulation may contain one or more permeation enhancers. Examples of suitable permeation enhancers may include, for example, mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium octoate, sodium caprate, sodium dodecyl sulfate, polyoxyethylene-9-lauryl ether, or EDTA.

In one embodiment, a frozen composition in frozen form is provided that contains a rAAV as described herein in a buffer solution. Optionally, one or more surfactants (e.g., pluronic) F68), stabilizers, or preservatives are present in the composition. Suitably, for use, the composition is thawed and titrated to the desired dose with a suitable diluent (e.g., sterile saline or buffered saline).

In certain embodiments, provided herein is a pharmaceutical composition comprising a vector (e.g., rAAV) described herein and a pharmaceutically acceptable carrier. As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Auxiliary active ingredients may also be incorporated into the composition. Delivery vehicles (e.g., liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc.) can be used to introduce the compositions of the invention into a suitable host cell. In particular, the rAAV vector can be formulated in a delivery form encapsulated in a lipid particle, liposome, vesicle, nanosphere, nanoparticle, or the like. In one embodiment, a therapeutically effective amount of the carrier is included in a pharmaceutical composition. The choice of carrier is not a limitation of the present invention. Other conventional pharmaceutically acceptable carriers, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerol, phenol, and p-chlorophenol. Suitable chemical stabilizers include gelatin and albumin.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.

As used herein, the term "dose" or "amount" may refer to the total dose or amount delivered to a subject during treatment, or the dose or amount delivered in a single unit (or multiple units or separate doses) administration.

Furthermore, the replication-defective virus composition may be formulated as a dosage unit containing replication-defective virus in an amount of about 1.0x10 ⁹ GC to about 1.0x10 ¹⁶ Gc (to treat subjects with an average body weight of 70 kg), including all integers or fractions within this range, is preferably 1.0x10 for human patients ¹² GC to 1.0x10 ¹⁴ And (3) GC. In one embodiment, the composition is formulated to contain at least 1x10 per dose ⁹ 、2x10 ⁹ 、3x10 ⁹ 、4x10 ⁹ 、5x10 ⁹ 、6x10 ⁹ 、7x10 ⁹ 、8x10 ⁹ Or 9x10 ⁹ GC, including all integer or fractional amounts within this range. In another embodiment, the composition is formulated to contain at least 1x10 per dose ¹⁰ 、2x10 ¹⁰ 、3x10 ¹⁰ 、4x10 ¹⁰ 、5x10 ¹⁰ 、6x10 ¹⁰ 、7x10 ¹⁰ 、8x10 ¹⁰ Or 9x10 ¹⁰ GC, including all integer or fractional amounts within this range. In another embodiment, the composition is formulated to contain at least 1x10 per dose ¹¹ 、2x10 ¹¹ 、3x10 ¹¹ 、4x10 ¹¹ 、5x10 ¹¹ 、6x10 ¹¹ 、7x10 ¹¹ 、8x10 ¹¹ Or 9x10 ¹¹ GC, including all integer or fractional amounts within this range. In another embodiment, the composition is formulated to contain at least 1x10 per dose ¹² 、2x10 ¹² 、3x10 ¹² 、4x10 ¹² 、5x10 ¹² 、6x10 ¹² 、7x10 ¹² 、8x10 ¹² Or 9x10 ¹² GC, including all integer or fractional amounts within this range. In another embodiment, the composition is formulated to contain at least 1x10 per dose ¹³ 、2x10 ¹³ 、3x10 ¹³ 、4x10 ¹³ 、5x10 ¹³ 、6x10 ¹³ 、7x10 ¹³ 、8x10 ¹³ Or 9x10 ¹³ GC, including all integer or fractional amounts within this range. In another embodiment, the composition is formulated to contain at least 1x10 per dose ¹⁴ 、2x10 ¹⁴ 、3x10 ¹⁴ 、4x10 ¹⁴ 、5x10 ¹⁴ 、6x10 ¹⁴ 、7x10 ¹⁴ 、8x10 ¹⁴ Or 9x10 ¹⁴ GC, including all integer or fractional amounts within this range. In another embodiment, the composition is formulated to contain at least 1x10 per dose ¹⁵ 、2x10 ¹⁵ 、3x10 ¹⁵ 、4x10 ¹⁵ 、5x10 ¹⁵ 、6x10 ¹⁵ 、7x10 ¹⁵ 、8x10 ¹⁵ Or 9x10 ¹⁵ GC, including all integer or fractional amounts within this range. In one embodiment, for human use, the dose may be 1x10 per dose ¹⁰ Up to about 1x10 ¹² GC, including all integer or fractional amounts within this range.

In certain embodiments, a pharmaceutical composition is provided that comprises a rAAV as described herein in a formulation buffer. In one embodiment, the rAAV is at about 1x10 ⁹ Genomic Copy (GC)/mL to about 1x10 ¹⁴ GC/mL formulation. In another embodiment, the rAAV is present at about 3x10 ⁹ GC/mL to about 3x10 ¹³ GC/mL formulation. In yet another embodiment, the rAAV is present at about 1x10 ⁹ GC/mL to about 1x10 ¹³ GC/mL formulation. In one embodiment, the rAAV is at least about 1x10 ¹¹ GC/mL formulation. In one embodiment, a pharmaceutical composition comprising a rAAV as described herein can be at about 1x10 ⁹ GC/gram brain mass to about 1x10 ¹⁴ GC/gram brain mass dose administration.

In certain embodiments, the compositions may be formulated in a suitable aqueous suspension medium (e.g., buffered saline) for delivery by any suitable route. The compositions provided herein are useful for systemic delivery of high doses of viral vectors. For rAAV, the high dose can be at least 1x10 ¹³ GC or at least 1x10 ¹⁴ And (3) GC. However, to increase safety, the miRNA sequences provided hereinThe columns may be contained in expression cassettes and/or vector genomes delivered at other lower doses.

The aqueous suspensions or pharmaceutical compositions described herein are designed for delivery to a subject in need thereof by any suitable route or combination of different routes. In one embodiment, the pharmaceutical composition is formulated for delivery by Intraventricular (ICV), intrathecal (IT), or intracisternal injection. In one embodiment, the compositions described herein are designed for delivery to a subject in need thereof by Intravenous (IV) injection. Alternatively, other routes of administration may be selected (e.g., oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intramuscular, and other parenteral routes). In certain embodiments, the compositions are delivered by two different routes substantially simultaneously.

As used herein, the term "intrathecal delivery" or "intrathecal administration" refers to a route of administration of a drug via injection into the spinal canal, more specifically into the subarachnoid space such that it reaches the cerebrospinal fluid (CSF). Intrathecal delivery may include lumbar puncture, intraventricular, suboccipital/intracisternal, and/or C1-2 puncture. For example, material may be introduced by lumbar puncture to diffuse throughout the subarachnoid space. In another example, the injection may enter the medullary canal of the cerebellum. Intracisternal delivery may increase carrier diffusion and/or reduce toxicity and inflammation caused by administration. See, e.g., christian Hinderer et al, broad gene transfer in the cynomolgus monkey central nervous system after delivery of AAV9 to the cisterna magna (Widespread gene transfer in the central nervous system of cynomolgus macaques following delivery of AAV9 into the cisterna magna), molecular therapy methods and clinical development (Mol Ther Methods Clin dev.)) (2014; 1:14051. on-line publication was made on day 12 and 10 of 2014. doi:10.1038/mtm.2014.51.

As used herein, the term "intracisternal delivery" or "intracisternal administration" refers to a route of administration by which a drug is administered directly into the cerebrospinal fluid of a ventricle or within a cerebellar medullary pool, more specifically by suboccipital puncture or by injection directly into the cerebellar medullary pool or by a permanently positioned tube.

It should be understood that the compositions of the pharmaceutical compositions described herein are intended to apply to the other compositions, protocols, aspects, embodiments, and methods described in the specification.

6. Therapeutic method

Provided herein are methods for fabry disease comprising delivering a therapeutically effective amount of a nucleic acid sequence or expression cassette comprising the hGLA coding sequence provided herein. In particular, the method comprises preventing, treating and/or alleviating the symptoms of fabry disease by delivering to a patient in need thereof a therapeutically effective amount of raav. In certain embodiments, a composition comprising an expression cassette described herein is administered to a subject in need thereof. In certain embodiments, the expression cassette is delivered by rAAV.

As used herein, "therapeutically effective amount" refers to the amount of the composition that delivers an amount of hGLA sufficient to alleviate or treat one or more symptoms of fabry disease. "treating" may include preventing exacerbations of the symptoms of fabry disease and possibly reversing one or more of its symptoms. A "therapeutically effective amount" of a human patient can be predicted based on an animal model. See, c.hinderer et al, molecular therapy (2014); 2212 2018-2027; bradbury et al, clinical development of human Gene therapy (Human Gene Therapy Clinical Development), 3 months 2015, 26 (1): 27-37, which are incorporated herein by reference.

In certain embodiments, treating comprises preventing, treating and/or alleviating one or more symptoms of fabry disease, including, for example, kidney disease, cardiomyopathy, pain, fatigue, stroke, hearing loss, gastrointestinal disorders.

In certain embodiments, the treatment comprises delivering an expression cassette, nucleic acid, vector (e.g., rAAV), or polypeptide described herein to one or more of the microvasculature, kidney cells, heart/heart cells, peripheral nerve, and cells of the central nervous system. In certain embodiments, the treatment results in a decrease in alpha-GalA substrate in one or more of cardiomyocytes, podocytes, vascular endothelial cells, and dorsal root ganglion. In certain embodiments, the treatment results in a decrease in alpha-GalA substrate in the kidney. In certain embodiments, the treatment results in a decrease in alpha-GalA substrate in the renal tubules.

In certain embodiments, the treatment comprises replacement or supplementation of defective alpha-galactosidase a in the patient by rAAV-based gene therapy. As expressed by the rAAV vectors described herein, expression levels of at least about 2% of the normal levels detected in CSF, serum, neurons, or other tissues or fluids may provide a therapeutic effect. However, higher expression levels may be achieved. Such expression levels may be 2% to about 100% of normal functional human GLA levels. In certain embodiments, higher than normal expression levels may be detected in serum or another biological fluid or tissue.

As used herein, the term "NAb titer" is a measure of how much neutralizing antibody (e.g., anti-AAV NAb) is produced that neutralizes the physiological effects of its targeting epitope (e.g., AAV). anti-AAV NAb titers can be found in Calcedo, r. Et al, (Worldwide Epidemiology of Neutralizing Antibodies to Adeno-Associated Viruses) global epidemiology of adeno-associated virus neutralizing antibodies, (journal of infectious diseases (Journal of Infectious Diseases), 2009). 199 (3): measurements were made as described in pages 381 through 390, which are incorporated herein by reference.

In certain embodiments, the compositions provided herein can be used to deliver a hGLA product of a desired function to a patient while inhibiting expression of a gene and/or gene product in dorsal root ganglion neurons. In certain embodiments, the method comprises delivering to the patient a composition comprising an expression cassette comprising an hGLA encoding sequence and a miRNA target sequence. In certain embodiments, the methods comprise delivering an expression cassette or vector genome comprising a miR-183 target sequence to inhibit the level of transgene expression in a DRG. In certain embodiments, the method comprises delivering an expression cassette for inhibiting transgene expression in a DRG, where the expression cassette comprises at least two miR183 target sequences, at least three miR183 target sequences, at least four miR183 target sequences, at least five miR183 target sequences, at least six miR183 target sequences, at least seven miR183 target sequences, or at least eight miR183 target sequences. In certain embodiments, the method comprises delivering an expression cassette for inhibiting transgene expression in a DRG, where the expression cassette comprises at least two miR182 target sequences, at least three miR182 target sequences, at least four miR182 target sequences, at least five miR182 target sequences, at least six miR182 target sequences, at least seven miR182 target sequences, or at least eight miR182 target sequences. In certain embodiments, the expression cassette comprises one or more miR182 target sequences and one or more miR183 target sequences.

Suitable delivery volumes of the provided compositions and their concentrations can be determined by one skilled in the art. For example, a volume of about 1 μl to 150mL may be selected, with a higher volume for adults. Typically, a suitable volume is about 0.5mL to about 10mL for newborn infants, and about 0.5mL to about 15mL for older infants may be selected. For young children, a volume of about 0.5mL to about 20mL may be selected. For children, a volume of up to about 30mL may be selected. For pre-pubertal and adolescents, volumes up to about 50mL may be selected. In other embodiments, the patient may receive intrathecal administration in a selected volume of about 5mL to about 15mL, or about 7.5mL to about 10 mL. Other suitable volumes and dosages may be determined. Dosages are adjusted to balance therapeutic benefit with any side effects, and such dosages may be varied depending upon the therapeutic application in which the recombinant vector is used.

In certain embodiments, compositions comprising a rAAV described herein can be at about 1x10 ⁹ GC/gram brain mass to about 1x10 ¹⁴ GC/gram brain mass dose administration. In certain embodiments, the rAAV is at about 1x10 ⁹ GC/kg body weight to about 1x10 ¹³ The GC/kg body weight dose is administered systemically.

In certain embodiments, the delivery of the expression cassette in the vector genome is about 1x10 ⁹ GC/gram brain mass to about 1x10 ¹³ Genome Copy (GC)/gram (g) brain mass, including all whole or fractional amounts within the range and endpoints. In another embodiment, the dose is 1x10 ¹⁰ GC/gram brain mass to about 1x10 ¹³ GC/gram brain mass. In particular embodiments, the carrier is administered to the patient at a dose of at least about 1.0x10 ⁹ GC/g, about 1.5x10 ⁹ GC/g, about 2.0x10 ⁹ GC/g, about 2.5x10 ⁹ GC/g, about 3.0x10 ⁹ GC/g, about 3.5x10 ⁹ GC/g, about 4.0x10 ⁹ GC/g, about 4.5x10 ⁹ GC/g, about 5.0x10 ⁹ GC/g, about 5.5x10 ⁹ GC/g, about 6.0x10 ⁹ GC/g, about 6.5x10 ⁹ GC/g, about 7.0x10 ⁹ GC/g, about 7.5x10 ⁹ GC/g, about 8.0x10 ⁹ GC/g, about 8.5x10 ⁹ GC/g, about 9.0x10 ⁹ GC/g, about 9.5x10 ⁹ GC/g, about 1.0x10 ¹⁰ GC/g, about 1.5x10 ¹⁰ GC/g, about 2.0x10 ¹⁰ GC/g, about 2.5x10 ¹⁰ GC/g, about 3.0x10 ¹⁰ GC/g, about 3.5x10 ¹⁰ GC/g, about 4.0x10 ¹⁰ GC/g, about 4.5x10 ¹⁰ GC/g, about 5.0x10 ¹⁰ GC/g, about 5.5x10 ¹⁰ GC/g, about 6.0x10 ¹⁰ GC/g, about 6.5x10 ¹⁰ GC/g, about 7.0x10 ¹⁰ GC/g, about 7.5x10 ¹⁰ GC/g, about 8.0x10 ¹⁰ GC/g, about 8.5x10 ¹⁰ GC/g, about 9.0x10 ¹⁰ GC/g, about 9.5x10 ¹⁰ GC/g, about 1.0x10 ¹¹ GC/g, about 1.5x10 ¹¹ GC/g, about 2.0x10 ¹¹ GC/g, about 2.5x10 ¹¹ GC/g, about 3.0x10 ¹¹ GC/g, about 3.5x10 ¹¹ GC/g, about 4.0x10 ¹¹ GC/g, about 4.5x10 ¹¹ GC/g, about 5.0x10 ¹¹ GC/g, about 5.5x10 ¹¹ GC/g, about 6.0x10 ¹¹ GC/g, about 6.5x10 ¹¹ GC/g, about 7.0x10 ¹¹ GC/g, about 7.5x10 ¹¹ GC/g, about 8.0x10 ¹¹ GC/g, about 8.5x10 ¹¹ GC/g, about 9.0x10 ¹¹ GC/g, about 9.5x10 ¹¹ GC/g, about 1.0x10 ¹² GC/g, about 1.5x10 ¹² GC/g, about 2.0x10 ¹² GC/g, about 2.5x10 ¹² GC/g, about 3.0x10 ¹² GC/g, about 3.5x10 ¹² GC/g, about 4.0x10 ¹² GC/g, about 4.5x10 ¹² GC/g, about 5.0x10 ¹² GC/g, about 5.5x10 ¹² GC/g, about 6.0x10 ¹² GC/g, about 6.5x10 ¹² GC/g, about 7.0x10 ¹² GC/g, about 7.5x10 ¹² GC/g, about 8.0x10 ¹² GC/g, about 8.5x10 ¹² GC/g, about 9.0x10 ¹² GC/g, about 9.5x10 ¹² GC/g, about 1.0x10 ¹³ GC/g, about 1.5x10 ¹³ GC/g, about 2.0x10 ¹³ GC/g, about 2.5x10 ¹³ GC/g, about 3.0x10 ¹³ GC/g, about 3.5x10 ¹³ GC/g, about 4.0x10 ¹³ GC/g, about 4.5x10 ¹³ GC/g, about 5.0x10 ¹³ GC/g, about 5.5x10 ¹³ GC/g, about 6.0x10 ¹³ GC/g, about 6.5x10 ¹³ GC/g, about 7.0x10 ¹³ GC/g, about 7.5x10 ¹³ GC/g, about 8.0x10 ¹³ GC/g, about 8.5x10 ¹³ GC/g, about 9.0x10 ¹³ GC/g, about 9.5x10 ¹³ GC/g or about 1.0x10 ¹⁴ GC/g brain mass.

In certain embodiments, the compositions provided herein are administered in combination with an immunosuppressant. Currently, immunosuppressants used in such combination therapies include, but are not limited to, glucocorticoids, steroids, antimetabolites, T-cell inhibitors, macrolides (e.g., rapamycin (rapamycin) or rapamycin analogs (rapalog)) and cytostatics, including alkylating agents, antimetabolites, cytotoxic antibiotics, antibodies or agents active against immunoaffinity. Immunosuppressants may include nitrogen mustard (nitrogenemustard), nitrosourea, platinum compounds, methotrexate (methotrexate), azathioprine (azathioprine), mercaptopurine (merapatopurine), fluorouracil (fluorouracil), actinomycin D (dactinomycin), anthracycline (anthracycline), mitomycin (mitomycin) C, bleomycin (bleomycin), mithramycin (mithramycin), IL-2 receptor- (CD 25-) or CD 3-directed antibodies, anti-IL-2 antibodies, cyclosporine (ciclosporin), tacrolimus (tacrolimus), sirolimus (sirolimus), IFN- β, IFN- γ, opioid (opid) or TNF- α binding agents. In certain embodiments, immunosuppressive therapy can begin 0, 1, 2, 7, or more days prior to administration of the gene therapy. Such therapy may include co-administration of two or more drugs (e.g., prednisone, mycophenolate Mofetil (MMF), and/or sirolimus (i.e., rapamycin)) on the same day. One or more of these drugs may be administered at the same dose or at a modified dose following administration of the gene therapy.

In certain embodiments, the rAAV provided herein is administered in combination with a therapy (combination therapy) such as an enzyme replacement therapy, a chaperone therapy, a substrate reduction therapy (e.g., sanofi-Genzyme and laneway (Idorsia)), and/or with a therapeutic (combination therapy) such as a microzyme therapy, a chaperone therapy, a substrate reduction therapy (e.g., sanofi-Genzyme) or a combination therapy (ldorsia)Antihistamines or other drugs that reduce the chance of infusion-related reactions are co-administered. In certain embodiments, the combination therapy is a functional hGLA protein (e.g.Sainofil-Jianzan; />Shire；/>ERT on plant basis) or a stable form of hGLA, as provided herein or as described in PCT/US2019/05567 (incorporated herein by reference) submitted on 10-2019. Administration may be oral or by intravenous infusion to an outpatient, and may include dosages suitable for daily, every other day, weekly, every two weeks (e.g., 0.2mg/kg body weight), monthly or bi-monthly administration. In certain embodiments, the combination therapy is a companion therapy (e.g., galafold (Mijistat, delivered orally in capsule form), aimei healthcare). The appropriate therapeutically effective dose for the combination treatment is selected by the treating clinician and includes about 1 μg/kg to about 500mg/kg, about 10mg/kg to about 100mg/kg, about 20mg/kg to about 100mg/kg, and about 20mg/kg to about 50mg/kg. In some embodiments, a suitable therapeutic dose is selected from, for example, 0.5, 0.75, 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 100, 150, 200, 250, 300, 400, or 500mg/kg.

In certain embodiments, a newborn infant (3 months of age or less) is treated according to the methods described herein. In certain embodiments, infants from 3 months of age to 9 months of age are treated according to the methods described herein. In certain embodiments, children 9 to 36 months of age are treated according to the methods described herein. In certain embodiments, children from 3 years to 12 years are treated according to the methods described herein. In certain embodiments, children from 12 years to 18 years are treated according to the methods described herein. In certain embodiments, an adult aged 18 years or older is treated according to the methods described herein.

In one embodiment, the patient suffering from fabry disease is a male or female having an age of at least about 3 months to less than 12 months. In another embodiment, the patient suffering from fabry disease is male or female, aged at least about 6 years up to 18 years. In other embodiments, the subject may be elderly or young, and may be male or female.

It should be understood that the compositions in the methods described herein are intended to apply to other compositions, protocols, aspects, embodiments, and methods described in the specification.

7. Kit for detecting a substance in a sample

In certain embodiments, a kit is provided that includes a concentrated carrier suspended in a formulation (optionally frozen), optionally a dilution buffer, and the devices and components required for intravenous, intrathecal, intraventricular, or intracisternal administration. In one embodiment, the kit provides sufficient buffer to allow injection. Such buffers may allow the concentrated carrier to be diluted in a ratio of about 1:1 to 1:5, or higher. Such kits may include additional non-carrier based active components wherein combination therapies and/or antihistamines, immunomodulators, etc. are used. In other embodiments, higher or lower amounts of buffer or sterile water are included to allow dose titration and other adjustments by the treating clinician. In other embodiments, one or more components of the device are included in a kit. Suitable dilution buffers are available, such as saline, phosphate Buffered Saline (PBS) or glycerol/PBS.

It will be appreciated that the compositions in the kits described herein are intended to apply to other compositions, protocols, aspects, embodiments, and methods described in the specification.

8. Device and method for controlling the same

In one aspect, the vectors provided herein may be intrathecally administered by methods and/or devices described, for example, in WO 2017/136500, which is incorporated herein by reference in its entirety. Alternatively, other apparatus and methods may be selected. In summary, the method comprises the steps of: advancing the spinal needle into a cerebral medullary canal of the patient, connecting a length of flexible tubing to a proximal hub of the spinal needle, and connecting an output port of the valve to a proximal end of the flexible tubing, and after the advancing and connecting steps and after allowing self-infusion with cerebrospinal fluid of the patient with the tubing, connecting a first container containing a quantity of isotonic solution to a flushing inlet of the valve, and then connecting a second container containing a quantity of pharmaceutical composition to a carrier inlet of the valve. After connecting the first and second containers to the valve, a fluid flow path between the carrier inlet and outlet of the valve is opened and the pharmaceutical composition is injected into the patient through the spinal needle, and after injecting the pharmaceutical composition, a fluid flow path through the irrigation inlet and outlet of the valve is opened and an isotonic solution is injected into the spinal needle to irrigate the pharmaceutical composition into the patient. The method and the device may each optionally be used for intrathecal delivery of the compositions provided herein. Alternatively, other methods and devices may be used for such intrathecal delivery.

It should be understood that the compositions in the devices described herein are intended to apply to other compositions, protocols, aspects, embodiments, and methods described in the specification.

Examples

The invention will now be described with reference to the following examples. These examples are provided for illustrative purposes only and the invention should in no way be construed as being limited to these examples, but rather should be construed to include any and all variations that become apparent as a result of the teachings provided herein.

Example 1: rAAHU68. HGLA for use in the treatment of Brillouin disease

The engineered sequence encoding hGLA was cloned into an expression construct containing CB7 promoter (a hybrid of the cytomegalovirus immediate early enhancer and the chicken β -actin promoter), chicken β -actin intron (CI), WPRE, and rabbit β -globin (rBG) polyadenylation sequences. The expression construct was flanked by AAV2 inverted terminal repeats and the AAVhu68 trans plasmid was used for encapsidation.

rAAV hu68.HGLA was generated by transfecting HEK293 cells with triple plasmids encoding the AAV cis plasmid flanking the AAV ITR transgene cassette, the AAV trans plasmid encoding the AAV2 rep and AAV hu68 cap genes (pAAV 2/hu68. KanR) and the helper adenovirus plasmid (pAdΔF6. KanR).

AAV cis plasmids

The map of the vector genome (SEQ ID NO: 6) is shown in FIG. 1. The vector genome contains the following sequence elements:

Inverted Terminal Repeat (ITR): ITR is the same reverse complement derived from AAV2 (130 bp, genBank: NC-001401), flanking all components of the vector genome. When AAV and adenovirus helper functions are provided in trans, the ITRs serve as origins of vector DNA replication and packaging signals for the vector genome. Thus, the ITR sequence represents the only cis sequence required for vector genome replication and packaging.

CB7 promoter: the promoter consists of a hybrid between the CMV IE enhancer and the chicken β -actin promoter.

Human cytomegalovirus immediate early (CMVIE) enhancer: this enhancer sequence obtained from human CMV (Gene Bank: K03104.1) increases the expression of downstream transgenes.

Chicken beta-actin (CB) promoter: such a ubiquitous promoter (GenBank: X00182.1) was chosen to drive transgene expression in any cell type.

Chicken beta-actin intron: the heterozygous intron consisted of a chicken beta-actin splice donor (973 bp, gene Bank: X00182.1) and a rabbit beta-globin splice acceptor element. Introns are transcribed but are removed from the mature mRNA by splicing, bringing together the sequences on either side of them. The presence of introns in the expression cassette has been shown to promote transport of mRNA from the nucleus to the cytoplasm, thereby enhancing the accumulation of stable levels of mRNA for translation. This is a common feature in gene vectors aimed at increasing the level of gene expression.

Coding sequence: an engineered cDNA (SEQ ID NO: 4) encoding hGLA (SEQ ID NO: 7) having cysteine residues (D233℃ I359C) (431 amino acids) at positions 233 and 359.

Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE): a cis-acting RNA element from Woodchuck Hepatitis Virus (WHV) was inserted into the 3' untranslated region of the coding sequence upstream of the polyA signal. WPRE is a sequence derived from hepadnavirus and has previously been used as a cis-acting regulatory module in viral gene vectors to achieve adequate levels of transgene product expression and to increase viral titres during manufacture. WPRE is thought to increase transgene product expression by improving transcription termination and enhancing 3' end transcriptional processing, thereby increasing the amount of polyadenylated transcript and the size of the polyA tail and resulting in more transgene mRNA available for translation. The WPRE contained in the cis plasmid was a mutant version containing five point mutations in the putative promoter region of the Open Reading Frame (ORF) of the woodchuck hepatitis virus X protein (WHX) protein and an additional point mutation (ATG mutated to TTG) in the start codon of the WHX protein ORF. Based on sensitive flow cytometry analysis of various human cell lines transduced with lentivirus containing a WPRE mut6-GFP fusion construct (Zanta-Boussif et al 2009), the mutant WPRE (termed mut 6) was considered sufficient to eliminate expression of the truncated WHX protein.

WPRE is a sequence derived from hepadnavirus and has previously been used as a cis-acting regulatory module in viral gene vectors to achieve adequate levels of transgene product expression and to increase viral titres during manufacture.

Rabbit β -globin polyadenylation signal (rBG PolyA): the rBG PolyA signal (127 bp, gene bank: V00882.1) promotes efficient polyadenylation of cis-transgenic mRNA. This element serves as a signal for transcription termination, a specific cleavage event occurs at the 3' end of the nascent transcript, and a long poly A tail is added.

B. Trans-plasmid: pAAV2/1.KanR (p 0068)

AAV2/hu68 trans plasmid is pAAV2/hu68.KanR (p 0068). The pAAV2/hu68.KanR plasmid is 8030bp in length and encodes four wild-type AAV2 replicase (Rep) proteins required for replication and packaging of the AAV vector genome. The pAAV2/hu68.Kanr plasmid also encodes three WTAAVhu68 virion protein capsid (Cap) proteins that assemble into the virion shell of AAV serotype hu68 to accommodate the AAV vector genome.

C. Adenovirus helper plasmid: pAdDeltaF6 (KanR)

The adenovirus helper plasmid pAdDeltaF6 (KanR) has a size of 15,770bp. The plasmid contains adenovirus genome regions important for AAV replication; namely, E2A, E4 and VA RNA (adenovirus E1 function is provided by HEK293 cells). However, plasmids do not contain other adenovirus replication or structural genes. Plasmids do not contain cis elements critical for replication, such as adenovirus ITRs; thus, it is expected that infectious adenoviruses will not be produced. Plasmids were derived from E1, E3 deleted molecular clones of Ad5 (pBHG 10, a pBR 322-based plasmid). Deletions were introduced into Ad5 to eliminate unnecessary expression of adenovirus genes and reduce the amount of adenovirus DNA from 32kb to 12kb. Finally, the ampicillin (ampicillin) resistance gene was replaced with a kanamycin (kanamycin) resistance gene to produce pAdeltaF6 (KanR). The E2, E4 and VAI adenovirus genes remaining in this plasmid and E1 present in HEK293 cells are essential for AAV vector production.

Example 2: method of

Transgene product expression-cell distribution

mRNA and protein localization was assessed in order to characterize distribution of transduction and transgene product expression following IV administration of AAVhu69.HGLA vector and correlate it with observed histological improvement of disease phenotype. Kidney, DRG and heart tissues were selected for evaluation as they are disease-associated target tissues for treatment of brile disease. Human GLA mRNA was assessed by In Situ Hybridization (ISH) in mice and NHPs. Human GLA protein was assessed in mice and NHPs by Immunohistochemistry (IHC) or immunofluorescence (1F). Sampling time points in mice and NHPs were selected to capture expression during the expected stable plateau of transgene product expression.

Expression of transgene product-functional Activity

To assess whether the transgene products observed by ISH and IHC are functional in mice and NHPs, GLA enzyme activity assays were performed. Kidney, heart, liver and DRG tissues are selected because they are disease-associated target tissues (kidney, heart) for treatment of brile disease and/or tissues that are readily transduced following IV gene therapy (liver, heart, DRG). Expression of the transgene product was not assessed in DRGs of mice due to their small size, while all other tissues were assessed in mice and NHPs. Sampling time points in mice and NHPs were selected to capture stable plateau transgene expression. GLA activity assays do not distinguish between human GLA transgene product and endogenous mouse or NHPGLA, so some background activity can be expected in untreated animals at baseline.

Thermal sensation function

The hotplate assay was performed because it measures thermal sensory defects in mice, which are believed to be similar to the tactile, painful and thermal sensory defects described in fabry patients secondary to DRG neuronal lysosomal storage and dysfunction. A decrease in latency response indicates an improvement in the fabry disease phenotype.

Renal function

BUN, urine osmotic pressure and urine volume were evaluated because they are biomarkers of kidney function. A decrease in BUN levels indicates an improvement in the fabry disease phenotype. An increase in urine osmotic pressure will indicate an increased ability to concentrate urine due to increased renal function, which represents an improvement in the fabry disease phenotype. A decrease in urine volume indicates an improvement in the fabry disease phenotype.

Lysosomal storage (GL-3 immunohistochemistry on tissues)

GLA enzyme deficiency results in accumulation of the toxic substrate GL-3 of the enzyme. Thus, GL-3 IHC was performed on DRG and kidneys, as they are organs that reproducibly showed significant storage in classical (Gla KO) and aggravated (Gla KO/TgG 3S) Fabry disease mouse models, and are target organs of pathology in Fabry disease patients (causing neuropathic pain and fatal renal failure, respectively). GL-3 IHC was performed on the heart because the aggravated (Gla KO/TgG S) Fabry-disease mice also showed storage in this organ. Reduced GL-3 storage indicates an improvement in the Fabry disease phenotype. GL-3 IHC sections were also stained with hematoxylin and eosin (H & E) to better visualize tissue morphology and detect potential adverse treatment-related findings.

Lysosomal storage (quantification of GL-3 and Lvso-Gb by LC-MS/MS ₃ )

Quantification of GL-3 storage in tissue by liquid chromatography-tandem mass spectrometry (LC-MS/MS), lyso-Gb in plasma or serum ₃ Quantification was performed. Since GL-3 is the primary substrate for the GLA enzyme and there is direct evidence that there is a relationship between the severity of substrate accumulation and the severity of Fabry disease, the quantification of storage in the target organ is performed. GL-3 storage reduction indicates legal clothImprovement of the lining phenotype.

Example 3: natural history study of exacerbation of Fabry disease (Gla KO/TgG 3S) and classical (Gla KO) mouse models

A natural history study was performed to characterize disease progression in a mouse model of fabry disease exacerbation (Gla KO/TgG 3S) and define the optimal pharmacological endpoint and treatment window for the efficacy study.

At birth, the study included 41 mice, including Gla WT males or Gla without TgG S allele ^+/- Heterozygous females (control; 5 males and 6 females), gla KO without TgG S allele (Gla) ^-/- The method comprises the steps of carrying out a first treatment on the surface of the 5 males and 5 females), gla WT male or Gla with one allele of TgG S ^+/- Heterozygote female (TgG 3S) ⁺ The method comprises the steps of carrying out a first treatment on the surface of the 5 males and 5 females) and Gla KO with one allele of TgG S (Gla KO/TgG S;5 males and 5 females). Body weight, hotplate performance, serum BUN levels, and urine osmotic pressure were periodically assessed. Necropsy was performed at 36 weeks of age, which is close to the published humane end-point of the model. Brain, spinal cord, DRG, heart, kidney, liver, skin, small intestine and large intestine were collected at necropsy for histological and assessment of GL-3 storage (GL-3 IHC and quantification by LC-MS/MS).

Study design: weighting Fabry Gla ^-/- /TgG3S ⁺ Natural history study in mouse model

^a Tissues were collected for evaluation of histopathology.

Abbreviations: BUN, blood urea nitrogen; gla, α -galactosidase a; tgG3S, human Gb3 synthase-transgenic.

All mice survived to the predetermined necropsy at week 36 except that two Gla KO/TgG3S mice were euthanized at weeks 33 and 35 due to disease-related weight loss.

Control, glaKO and TgG S mice increased body weight at each time point throughout the study (fig. 10A and 10B). In contrast, glaKO/TgG3S mice reached a peak in body weight at 18 weeks (24.4 g for males and 21.5g for females), after which the mice began to lose body weight until necropsy.

Throughout the study, male and female TgG S mice exhibited a similar hotplate latency to sex matched control animals, indicating a normal sensory response. From 25 weeks of age to the end of the study, male and female GlaKO mice exhibited slightly longer dry soaking latency, indicating a slightly reduced sensory response, compared to the sex-matched controls. In contrast, from 25 weeks of age to the end of the study, male GlaKO/TgG3S mice exhibited significantly longer average latency responses than control or GlaKO mice (male or female), indicating that male GlaKO/TgG S mice had more severe sensory deficits than male and female GlaKO mice. Female GlaKO/TgG3S mice also showed slightly longer hotplate latency responses than control mice, but the latency was similar to female GlaKO mice, indicating similar sensory deficit in both female fabry disease mouse models (fig. 11A and 11B).

Throughout the study, male and female TgG S and Gla KO mice exhibited similar serum BUN levels to their sex-matched controls, indicating normal kidney function. In contrast, both 25 week old male and female Gla KO/TgG3S mice exhibited elevated BUN levels compared to sex matched controls. During the course of the study, BUN levels in males and females generally increased, indicating reduced renal function. BUN levels were approximately similar in male and female Gla KO/TgG S mice throughout the study (FIGS. 12A and 12B).

In vivo variability in urine osmotic pressure was observed in the study. However, from 25 weeks of age until the end of the study, male and female Gla KO/TgG S mice generally exhibited lower mean urine osmotic pressure than gender matched control and Gla KO mice, indicating failure to concentrate urine due to reduced renal function (fig. 13A and 13B).

When assessed by IHC, more significant storage of GL-3 in the kidney and secondary injury to nephritis and tubular necrosis was observed in male Gla KO/TgG S mice compared to Gla KO or WT/TgG S mice (fig. 14A). GL-3 storage and secondary pathology was greater in Gla KO/TgG3S mice than in Gla KO mice. In the kidneys of Gla KO/TgG3S mice, storage material was observed in both the tubules and glomerular cells, whereas GL-3 storage was observed only in the tubules of Gla KO mice. In addition to more storage in the tubules and glomeruli, some Gla KO/TgG S3S mice exhibited secondary inflammatory and degenerative changes in the kidneys (tubular degeneration, necrosis and secondary interstitial mononuclear nephritis), which were not seen in any Gla KO mice. Hearts did not exhibit any GL-3 storage or secondary injury in Gla KO mice, and in some Gla KO/TgG3S animals, some GL-3 storage material was exhibited in cardiac myocytes as well as myocardial cell necrosis and mineralization.

Quantification of GL-3 IHC demonstrated that male Gla KO/TgG S mice had significantly higher levels of GL-3 storage in the whole kidney compared to either Gla KO or WT/TgG S mice, with WT/TgG3S mice having the lowest levels of GL-3 storage in the 3 mouse models (FIG. 14B).

When assessed by IHC, male Gla KO/TgG3S mice exhibited significant GL-3 storage in DRG sensory neurons (FIG. 15A). Although male Gla KO mice also exhibited GL-3 storage in DRG sensory neurons, very little DRG GL-3 storage was observed in male WT/TgGS3 mice. Quantification of IHC staining showed a significant increase in DRG GL-3 storage in Gla KO/TgG S mice compared to Gla KO or WT/TgG3S models, WT/TgG3S mice exhibited minimal levels of DRG GL-3 storage (FIG. 15B).

The substrate (lyso-Gb in plasma) was purified by LC-MS/MS ₃ Quantification of GL-3) in tissues demonstrated that these substrates were stored more in kidneys, heart, brain and plasma of the aggravated mice (Gla KO/TgG 3S) than Gla KO mice (fig. 16A to 16D). In the kidneys of male wild-type mice, GL-3 storage was very low, allowing a slight increase in GL-3 storage in TgG S mice to be distinguished. GL-3 storage in male Gla KO mice was greater than in TgG S mice, and the aggravated Gla KO/TgG S mice exhibited significantly increased GL-3 storage compared to the levels observed in Gla KO mice. GL-3 stores in female mouse kidney tissue indicated a clear trend of GL-3 stores increasing from wild-type mice with minimal levels, followed by TgG S and Gla KO mice, with Gla KO/TgG S mice having minimal levels High storage levels.

In heart tissue of male animals, there was minimal GL-3 storage in wild type and TgG S mice. Although GLA KO male mice had slightly more GL-3 storage in heart tissue, the aggravated GLA KO/TgG3S mice had significantly increased substrate storage levels. In female mice, there was minimal GL-3 storage in heart tissue of wild-type mice, with slightly increased levels in TgG S and Gla KO mice, and significantly increased GL-3 storage was observed in Gla KO/TgG S mice.

In brain tissue of male and female mice, GL-3 storage levels were lower in wild-type, gla KO and TgG S mice. In both sexes, gla KO/TgG3S mice had significantly increased GL-3 storage in the brain compared to the other three models studied.

In plasma, the level of lyso-Gb3 storage was minimal in both wild-type and TgG3S mice. These levels increased to a similar extent in male Gla KO and Gla KO/TgG3S mice. In female mice, the level of lyso-Gb3 storage in Gla KO mice was increased compared to wild-type and TgG S models; however, the level of lyso-Gb3 storage was significantly increased between Gla KO and Gla KO/TgG3S mice.

Cumulatively, this natural history study demonstrated that the worsening fabry disease mouse model (Gla KO/TgG3S mice) over-expressed human Gb3 synthase began to develop disease-related abnormalities at about 18 to 25 weeks of age (4.5 to 6 months of age). In addition, gla KO/TgG3S mice generally exhibit a more severe phenotype than non-aggravated fabry disease mice (Gla KO). Specifically, gla KO/TgG3S mice exhibited more severe weight loss (emaciation [ male and female ]) and sensory deficit (increased hotplate latency [ male only ]) compared to gender matched Gla KO mice. Gla KO/TgG3S mice also showed progressive kidney injury (increased serum BUN levels, decreased urinary osmotic pressure [ male and female ]), which was not apparent in Gla KO mice, and further showed greater accumulation of GL-3 in kidneys, heart, DRG, brain and plasma. The aggravated fabry disease mouse model (Gla KO/TgG 3S) also demonstrated some secondary degenerative, necrotic and mineralized lesions never observed in the kidneys (mononuclear degenerative interstitial nephritis) and heart (cardiomyocyte necrosis and mineralization), and possibly explained a more pronounced phenotype. Interestingly, storage material, including podocytes, was also observed in the glomeruli, similar to fabry patients and different from Gla KO mice. Storage in podocytes (cells constituting the filtration barrier in the glomeruli) is critical to the pathophysiology of fabry disease and may explain the increased proteinuria and decreased urine osmotic pressure in mouse exacerbation models and patients.

GLA KO/TgG3S mice develop progressive ataxia with severe tremor and ambulatory deficit, causing euthanasia around 35 to 40 weeks of age, unlike GLA KO mice which exhibit normal longevity. This appears to be attributable to significant GL-3 stores in the CNS, including in the cerebellum where Purkinje cells (Purkinje cells) were histologically observed to denature and lose. However, purkinje cell degeneration and ataxia are not a characteristic of human fabry disease. In the mouse model of exacerbation, artificial overload of the Gla substrate GL-3 was achieved by overexpression of GL-3 synthase driven by a ubiquitous promoter. Accumulation of GL-3 in the CNS is continuous with neuronal overexpression of Gb3S in the absence of GLA in the double mutant Gla KO/TgG S. Thus, mouse ataxia is directly attributed to a broad and prominent Gb3 storage material in the CNS, and it can be alleviated by gene therapy that will restore GLA levels. For this reason, the monitoring of ataxia and survival in a model of a aggravated mouse is a relevant biomarker for the mouse, even if it is not a translated biomarker.

In summary, the results of this study support the use of the aggravated fabry disease Gla KO/TgG3S mouse model as a test system for efficacy studies with the following efficacy endpoints: lyso-Gb3 storage in plasma, GL-3 storage in tissues (kidney, heart, DRG, brain), histopathology (kidney, heart, DRG, brain), thermo-sensory function (hotplate), renal function (BUN, urine osmotic pressure), ataxia and survival.

Example 4: assessment of rAAV vectors for delivery of hGLA for gene therapy

The aim of this study was to determine the optimal human alpha-galactosidase a (hGLA) amino acid sequence for gene therapy. Constructs encoding hGLA variants were tested. For fair comparison, the vector included the same capsid and promoter, and the WPRE enhancer was also present in all expression cassettes.

Two to three month old fabry disease mice (Gla KO) were injected Intravenously (IV) with various aavhu68.Hgla vectors at one of the following doses: 1x10 ¹¹ GC(5x10 ¹² GC/kg-medium dose) or 5X10 ¹¹ GC(2.5x10 ¹³ GC/kg-high dose). PBS-treated Fabry disease Gla KO and WT mice served as controls. Blood was collected for serum separation at 1 week and 3 weeks after injection (pi) and blood was collected for plasma separation at 4 weeks (necropsy time point) of pi. Brains, spinal cords with Dorsal Root Ganglion (DRG), heart, kidneys, liver, skin, small intestine and large intestine were collected at pi 4 weeks, half of which were treated for histology and half were frozen for biochemical analysis (storage quantification by quantitative mass spectrometry and GLA enzyme activity measurement). The primary efficacy endpoint for the comparative vehicle is the quantification of the stored material in the target organ. In Gla KO mice, the storage material acyl sphingosine trihexose (GL-3) can be stained by Immunohistochemistry (IHC) on zinc-formalin paraffin-embedded tissue sections. Brown deposits are seen in tubular epithelial cells, with progressive deterioration of storage with age. Can also be stored in H &The E-stained DRG neurons appear as enlarged, transparent stained neurons (their transparent color is due to glycolipid storage material in the cytoplasm). Other target organs of fabry disease (such as heart, intestine or brain vasculature) showed low and inconsistent storage staining in the traditional Gla KO mouse model. These organs were collected and processed, but no efficacy assessment of the vector was allowed.

Gla KO mice are a model widely used for Fabry disease (Ohshima T, murray GJ, swaim WD, longnecker G, quirk JM, cardarelli CO, sugimoto Y, pastan I, gottesman MM, brady RO, kulkarni AB:. Alpha. -galactosidase A deficient mice: fabry disease model (. Alpha. -Galactosidase A deficient mice: A model ofFabry disease),. Alpha. -national academy of sciences (Proc Natl Acad Sci) 94:2540-2544, 1997). Hemizygous males exhibit abnormal kidney and liver morphology, both with accumulation of acyl sheath saddle trihexoses. They also exhibit mild cardiomyopathy and abnormal cardiovascular physiology. The small size, reproducible phenotype and efficient breeding allow for rapid studies that are optimal for in vivo screening of vectors in the clinic.

The following carriers are hinged:

AAVhu68.CB7.hGLAnat.WPRE.rBG

AAVhu68.CB7.hGLAco.WPRE.rBG

AAVhu68.CB7.hGLAco(M51C_G360C).rBG

the IV pathway was chosen for ease of performance, reproducibility and robust liver and heart transduction, allowing the extraction of transgenic GLA for analysis. It is also the intended clinical route of administration. Selecting a selected dose range of 1x10 ¹¹ GC to 5X10 ¹¹ GC (equivalent to about 5X10 ¹² GC/kg to 2.5x10 ¹³ GC/kg) to achieve muscle, heart and liver transduction at the highest dose. The lowest dose is expected to be suboptimal and thus better distinguishes the therapeutic effect between different carriers.

Each group included a minimum of 6 mice (male and female) to enable statistical analysis of pharmacological readings.

Pharmacological readings include biochemical assays (including but not necessarily limited to measurement of GLA enzyme activity, total enzyme amount, binding to mannose-6-phosphate receptor, GL-3 storage) and histological endpoints (GL-3 staining). Antibodies to hGLA were determined.

List of group names

Results

GLA activity levels measured in serum samples obtained 1 week post injection showed that GLA activity water-dried as a whole was dose dependent, with a concentration of GLA at 2.5x10 ¹³ GC/kg dose observations on all three vectorsTo a higher level (fig. 17). All three vectors produced higher levels of GLA activity compared to wild-type and GLAKO controls. In the higher dose group (2.5x10 ¹³ GC/kg), mice administered with AAVhu68. HGLASA and AAVhu68. HGLASO exhibited higher levels of GLA activity than mice injected with AAVhu68. HGLASO (M51C_G360C). Male mice exhibited higher enzymatic activity than female mice, as expected, due to more efficient AAV transduction and gene expression in hepatocytes from males than female mice, a mouse-specific phenomenon not encountered in non-human primates and humans. At the administration of two doses of AAVhu68.HGLACO (M51C_G360C) and a low dose (5.0x10) ¹² GC/kg) Male mice of AAVhu68. HGLASAT and AAVhu68. HGLASO were similar in GLA activity. However, after injection of high doses (2.5x10 ¹³ GC/kg) AAVhu68. HGLASAT and AAVhu68.HGLACO were significantly higher in GLA activity levels. Although the GLA activity level in female mice was much lower, the same trend in GLA activity levels in the three vectors observed in male mice was also observed in female mice.

The majority of enzyme activity measured in serum is derived from proteins expressed and secreted in hepatocytes. To investigate the reason for the lower enzyme activity in serum observed with the vector expressing the engineered candidate aavhu68.Hglaco (m51c—g360C), we performed vector genomic biodistribution analysis in liver samples collected at necropsy. For all three vectors, a higher dose (2.5x10 ¹³ GC/kg) showed higher transduction rates than mice injected with the corresponding lower doses of each vector, and 3 different vectors produced similar levels of vector genome at the given dose level indicating that the reduced enzymatic activity of the vector encoding the engineered candidate was attributable to reduced expression of the transgene (fig. 18).

Also at IV injection 5.0x10 ¹² Or 2.5x10 ¹³ The level of cathepsin activity in disease-related organs was analyzed on day 28 after GC/kg dose of vector. The lower graph shows the results of enzymatic activity of heart (fig. 19), liver (fig. 20), kidney (fig. 21), brain (fig. 22) and small intestine (fig. 23). After administration of high doses (2.5x10 ¹³ GC/kg) AAVhu68.HGLANat and AAVhu68.HGLACO,the total GLA activity level measured in heart tissue was highest. After administration of low doses (5.0x10 ¹² GC/kg) and AAVhu68. HGLASO, and two doses of AAVhu68.HGLA activity levels were observed in mice with AAVhu68. HGLASO (M51C_G360C). Similar activity levels were observed in both male and female mice.

In the liver, overall GLA activity was demonstrated in the administration of high doses (2.5x10 ¹³ GC/kg) was higher in all three AAVhu68.HGLA mice and highest in mice treated with AAVhu68.HGLANat and AAVhu68. HGLACO. In general, GLA activity was slightly higher in male mice compared to female mice.

Although there was less dose-dependent change in overall GLA activity measured in kidney tissue, the dose was measured at the high dose (2.5x10 ¹³ GC/kg) the activity level in all three aavhu68.Hgla mice still tended to be higher. There was no significant difference in GLA activity levels observed between male and female mice; however, GLA activity levels measured in female mice were more variable.

GLA activity was observed in brain tissue of wild-type mice with significant water dryness. Similarly, GLA active water dry was used in the administration of high doses (2.5x10 ¹³ GC/kg) was higher in all three AAVhu68.HGLA mice and was found to be higher in mice with high doses (2.5x10 ¹³ GC/kg) were highest among AAVhu68.HGLANat and AAVhu68.HGLaco treated mice. Similar activity levels were observed in both male and female mice.

Two doses of AAVhu68.HGLACO (M51C_G360C) and a low dose (5.0x10) ¹² GC/kg) of AAVhu68. HGLNAt and AAVhu68.HGLACo treated mice had lower GLA activity levels in the small intestine and were similar in magnitude. In mice at high dose (2.5x10 ¹³ GC/kg) of GLA activity was observed at the highest level in AAVhu68.HGLANat and AAVhu68. HGLaco. No significant changes were observed between male and female mice.

In summary, consistent with the serum results described above, the tissue level of GLA enzyme activity was dose-dependent, comparable between the two candidates encoding the unmodified native protein (engineered or unengineered) and significantly lower in the candidate encoding the engineered protein hGLAco (m51c—g360C). No gender effect was observed in organs other than the liver.

To further evaluate the pharmacology of these three vectors, we measured the amounts of stored material lyso-Gb3 and GL-3 in tissues in plasma from GLA KO mice by LC-MS/MS 28 days after AAV administration and compared these levels to those measured in PBS-treated GLA KO and wild-type control mice. Reduced Lyso-Gb3 and GL-3 stores are consistent with enzyme activity levels; two vectors encoding GLA (aavhu 68.Hglanat and aavhu68. Hglaco) resulted in high doses (2.5x10) in plasma, renal and cardiac samples ¹³ Complete storage of GC/kg was eliminated (FIG. 24). However, the vector encoding hGLACO (M51C_G360C) only partially reduced the level of lyso-Gb3 storage in plasma and GL-3 storage in kidney and heart tissue.

Example 5: assessment of rAAV vectors for delivery of hGLA for gene therapy

The purpose of this study was to evaluate up to three different doses (2.5x10 ¹² GC/kg，5x10 ¹² GC/kg，2.5x10 ¹³ GC/kg) to determine the efficacy in Gla KO mice after IV administration. All vectors evaluated have the same capsid, promoter and polyA signal, but include different versions of the human GLA transgene. The three transgenes evaluated were hGALCo (same as in example 4), hGALCo (M51C_G360C) (same as in example 4), and hGALA-D233C-1359 Cco. The hGLAco (m51c—g360C) transgene encodes an engineered GLA protein with two point mutations that introduce disulfide bonds to stabilize the enzyme in its active dimeric form. The hGLAco (d233c_i359C) transgene encodes a second version of the engineered GLA protein with two point mutations that introduce disulfide bonds to stabilize the enzyme in its active dimeric form.

Adult mice (3.5 to 4.5 months of age) received a single IV administration of 1 of 3 candidate vectors (aavhu 68.Hglaco, aavhu68.Hglaco (m51c_g360C) or aavhu68.Hglaco (d233c_i359C), at a low dose of 2.5x10 ¹² GC/kg, medium dose 5.0x10 ¹² GC/kg, or high dose of 2.5x10 ¹³ GC/kg (AAVhu68. HGLACO only (D233C-I359C)). Vehicle (PBS) -treated WT and Gla KO mice served as controls.

Animals were monitored daily for viability. Serum was collected on day 7 to assess transgene product expression (GLA enzyme activity). On day 28, necropsy was performed, heart, kidney, liver and spinal cord with DRG were collected and processed for histological, GL-3 quantification and assessment of GLA enzyme activity. Plasma was also collected for lyso-Gb ₃ GLA enzyme activity was quantified and assessed.

Intravenous administration was well tolerated by all the vectors evaluated. All animals survived to the predetermined necropsy time point.

In summary data of male and female Gla KO mice, the administration of high doses (2.5x10 ¹³ GC/kg) Gla KO mice of AAVhu68.HGLaco (D233C_I 359C), the serum transgene product expression (GLA enzyme activity) was highest. GLA enzyme activity levels were similar in GLA KO mice in the remaining treatment groups, although there was a slight dose-dependent response in GLA KO mice administered with aavhu68.Hglaco or aavhu68.Hglaco (d233 c_i 359C). Male Gla KO mice exhibited higher GLA enzyme activity than females. After administration of high doses (2.5x10 ¹³ GC/kg) AAVhu68.HGLaco (D233C_I 359C) had the highest GLA enzyme activity, and there was a dose-dependent GLA enzyme activity in the male Gla KO mice administered with AAVhu68.HGLaco or AAVhu68.HGLaco (D233C_I 359C). Gla enzyme activity in female Gla KO mice was very low, at high doses (2.5x10 ¹³ The highest level was observed in mice of AAVhu68.HGLaco (D233 C_I 359C) (FIG. 25).

Summary data of transgene product expression (GLA enzyme activity) measured in plasma collected 28 days post-administration revealed a clear dose-dependent effect of all 3 AAV vectors studied, with the dose (5.0x10 during administration ¹² GC/kg) AAVhu68.HGLaco and high dose (2.5x10) ¹³ GC/kg) of AAVhu68.HGLACO (D233C_I 359C) of Gla KO mice, the highest level of GLA enzyme activity was observed. GLA enzyme activity was much higher in male GLA KO mice than in female GLA KO mice. The dose-dependent effect of aavhu68.Hgla on Gla activity of all samples was observed in male Gla KO mice, with a medium dose (5.0x10 ¹² GC/kg) AAVhu68.HGLaco and high dose (2.5x10) ¹³ GC/kg) AAVhu68.HGLaco (D233 C_I 359C) gave the highest enzyme activity. Gla activity levels were generally lower in female Gla KO mice, high dose (2.5x10 ¹³ GC/kg) AAVhu68.HGLaco (D233 C_I 359C) provided the highest level of activity (FIG. 26).

Fig. 27, 28 and 29 show GLA enzyme activity in heart, liver and kidney tissue, respectively.

Summary data for GLA enzyme activity in cardiac tissue showed that all 3 AAV vectors studied had significant dose-dependent effects, with the dose (5.0x10 during administration ¹² GC/kg) AAVhu68.HGLaco and high dose (2.5x10) ¹³ GC/kg) of AAVhu68.HGLACO (D233C_I 359C) of Gla KO mice, the highest level of GLA enzyme activity was observed. Similar levels of Gla enzyme activity were observed in male and female Gla KO mice.

Comprehensive data for GLA enzyme activity in liver samples revealed that two doses of aavhu68.Hglaco and a high dose (2.5x10) were administered ¹³ GC/kg) AAVhu68.HGLaco (D233C_I 359C) Gla KO mice had the highest level of enzyme activity. These observations are reflected in the results of male Gla KO mice. Female Gla KO mice have significantly lower levels of Gla enzyme activity than male Gla KO mice and exhibit smaller activity differences between the three AAV vectors and dose.

Gla enzyme activity measured in kidney samples showed a clear dose-dependent effect on all 3 aavhu68.Hgla vectors studied in male and female Gla KO mice, at the dose (5.0x10 ¹² GC/kg) AAVhu68.HGLACO and AAVhu68.HGLACO (M51C_G360C) and high dose (2.5x10) ¹³ GC/kg) had the highest level of activity in the AAVhu68.HGLaco (D233 C_I 359C) mice. These trends are reflected when GLA enzyme activity is analyzed by sex, which also reveals similar GLA enzyme activity levels in male and female GLA KO mice.

Plasma from treated Gla KO mice was evaluated to evaluate the carrier in decreasing lyso-Gb ₃ Curative effect on storage (fig. 30). Medium dose for data display (5.0x10) ¹² GC/kg) AAVhu68.HGLaco and medium and high doses (5.0x10, respectively) ¹² GC/kg and 2.5x10 ¹³ GC/kg) AAVhu68.HGLaco (D233C_I 359C) treated Gla KO mice were completely depleted of lyso-Gb in plasma ₃ And (5) storing. These results were consistent in both male and female Gla KO mice.

Immunohistochemical data from kidney tissue samples revealed that while some reduction in GL-3 storage was observed at the highest dose where all three vectors were administered, at the high dose (2.5x10 ¹³ The most significant reduction in GL-3 storage was observed in the Gla KO mice treated with AAVhu68.HGLaco (D233C_I 359C) (FIG. 31A). Consistent with previous results, quantification of kidney IHC-stained GL-3 stores revealed that Gla KO mice treated with all 3 doses of AAVhu68.HGLaco (D233C-I359C) had significantly less GL-3 stores in the kidney tubules than vehicle-treated Gla KO controls. None of the mice treated with aavhu68.Hglaco or other engineered variants aavhu68.Hglaco (m51c—g360C) had significant reduction in storage. This reduction in GL-3 storage was observed to be dose dependent upon administration of the highest dose (2.5x10 ¹³ GC/kg) had the greatest effect in Gla KO mice of AAVhu68.HGLaco (D233C_I 359C) (FIG. 31B).

Immunohistochemical data from DRG longitudinal sections showed minimal GL-3 storage in Gla KO mice treated with aavhu68.Hglaco (D233 c_i 359C) compared to Gla KO mice treated with vehicle, aavhu68.Hglaco or aavhu68.Hglaco (m51c_g360C) (fig. 32A). Quantification of these IHC data showed a significant reduction in DRG neuron GL-3 storage in Gla KO mice treated with all three doses of aavhu68.Hglaco (d233c_1359c) compared to Gla KO mice treated with vehicle. This response was observed to be dose dependent upon administration of the highest dose (2.5x10 ¹³ GC/kg) had the greatest effect in the AAVhu68.HGLaco (D233C_I 359C) mice. Likewise, other candidates aavhu68.Hglaco and other engineered variants aavhu68.Hglaco (m51c—g360C) did not result in significant GL-3 storage reduction in DRG (fig. 32B).

Cumulatively, administration of aavhu68.Hglaco (d233c_i359C) to Gla KO mice resulted in significant transgene product expression (Gla enzyme activity), with the highest plasma and tissue in vivo efficacy compared to the other vectors evaluated. Aavhu68.Hglaco (d233 c_i359C) treated Gla KO mice exhibited a significant dose-dependent reduction in kidney and DRG GL-3 storage compared to vehicle treated Gla KO mice, whereas administration of non-engineered aavhu68.Hglaco vector did not significantly reduce GL-3 storage at the same dose level.

Example 6: evaluation of AAVhu68.HGLACO (D233 C_I 359C) in non-human primate

The present study was aimed at evaluating the primary pharmacology and safety of intravenous administration of aavhu68.Hglaco (d233 c_i 359C) to cynomolgus monkeys.

Adult NHP (n=4) received a single IV dose of aavhu68.Hglaco (d233 c_i 359C), at a dose of 2.5x10 ¹³ GC/kg. The in vivo evaluation included daily clinical observations, body weight, blood clinical pathology (CBC, coagulation panel, serum chemistry [ including cardiac biomarker troponin I ] ]) And cardiac function assessment (EKG and echocardiography). All animals were necropsied at day 60±3 after vehicle administration. At necropsy, tissues were collected for histopathological examination. Target tissues were collected for vector biodistribution analysis and transgene product expression localization (human GLA ISH mRNA]And human GLAIHC [ protein ]]) Is a comprehensive histological evaluation of (c). PBMCs, splenocytes and hepatic lymphocytes were also collected to measure T cell responses to capsid and transgene products (IFN- γ ELIspot). The study design is shown in the following table.

^a BL was up to 21 days prior to dosing.

^b Serum chemistry analysis included troponin I (cardiac biomarker).

^c Samples were collected and stored for future analysis.

^d Tissue was collected for histopathology, vector biodistribution analysis, and transgene product expression (human GLA ISH staining [ mRNA]And person GLA IHC staining [ protein]) Is a histological evaluation of (c). Liver and spleen lymphocytes were collected for measuring T cell responses to capsid or transgene products (IFN- γ ELISpot).

Abbreviations: ADA, anti-drug antibody; BL, baseline; ELISPot, enzyme-linked immunosorbent assay; GLA, α -galactosidase; IFN-gamma, interferon gamma; IHC, immunohistochemistry; IN, intranasal; ISH, in situ hybridization; mRNA, messenger ribonucleic acid; MS, mass spectrometry; nab, neutralizing antibodies; PBMC, peripheral blood mononuclear cells

Baseline sample collection:

baseline blood samples including Complete Blood Counts (CBC), clotting, cardiac biomarkers, serum chemistry, and PBMC/ELISPOT were collected from all animals up to 21 days prior to administration of the test or control (baseline) and at time points shown in the table below. Vital signs (i.e., temperature, heart rate, respiration) were obtained from each animal prior to sample collection.

a) Capsid neutralizing antibodies (serum): blood (up to 2 mL) for detecting the presence of AAVhu68 Nab was collected at baseline and day 60 (necropsy). Blood was collected through a red-top tube (with or without serum separator), allowed to coagulate, and centrifuged.

b) Cardiac biomarker (serum): blood (up to 2 mL) for detecting the presence of a cardiac toxicity marker (troponin I) was collected at baseline, day 3, day 7, day 14, day 28 and day 60 (necropsy). Blood was collected through a red-top tube (with or without serum separator), allowed to coagulate, and centrifuged. Serum was isolated.

c) Transgene expression, antibodies, complement factors or cytokines (plasma): blood (at least 3 mL) for detecting hGLA, anti-hGLAAb and/or complement activation or cytokine (in case of toxicity) presence was collected on day 0, day 3, day 7, day 14, day 28 and day 60 (necropsy). Blood was collected in a labeled light purple cap (EDTAK 2) and centrifuged at about 2700RPM (1300.+ -. 100x g) in a centrifuge at about +4 ℃ for 15 minutes within 30 minutes after collection.

d) PBMC/ELISPOT: blood (5 to 10 mL) was collected into heparin sodium (green top tube) and PBMCs were isolated. Samples were collected at baseline and necropsy. T cell responses to capsids and/or transgenes were assessed.

e) Hematology (cell count and classification): blood (up to 2 mL) was collected to obtain a whole blood count with differential and platelet counts. The following parameters were analyzed at specific time points specified in the study design:

erythrocyte count

Hemoglobin (hemoglobin)

Hematocrit

Average erythrocyte volume (MCV)

Mean erythrocyte hemoglobin (MCH)

Mean erythrocyte hemoglobin concentration (MCHC)

Platelet count

White blood cell count

Leukocyte classification

Erythrocyte morphology (pathologist examination)

Reticulocyte count

f) Clinical chemistry: blood (up to 2.0 mL) for clinical chemistry studies was collected in labeled red-top (serum) tubes, allowed to clot for up to 15 minutes, and centrifuged. Serum was isolated and placed in a labeled microcentrifuge tube. The following parameters were analyzed at specific time points specified in the study design:

alkaline phosphatase

Hemolysis marker: bilirubin (direct, indirect)

Creatinine

Gamma-glutamyl transpeptidase

Glucose

Serum alanine aminotransferase

Serum aspartate aminotransferase

Albumin

Albumin/globulin ratio (calculated)

Blood urea nitrogen

g) Coagulation: blood (2.0 mL) for the clotting panel was collected in a labeled blue top (citrate) tube. The following parameters were analyzed at specific time points specified in the study design:

PTT

PT

fibrinogen

D-dimer

Fibrin degradation products

Cardiac monitoring

The study involved baseline echocardiography prior to vehicle administration and additional echoes at study termination. Minimum parameters assessed include diastolic/systolic volume, stroke volume, cardiac output, fractional shortening, septum thickness, ejection time. Ejection fraction of the apex two or four chambers, right parasternal long axis four chamber view, right parasternal short axis view was also evaluated, when combined with heart rate, yielding left ventricular ejection fraction, end diastole volume, end systole volume, stroke volume, and cardiac output.

Results:

based on clinical observations, intravenous administration was well tolerated and all animals survived to the predetermined necropsy time point. At baseline, day 14, day 28 and day 60, troponin I levels (indicative of myocardial cell injury) were below the reportable range of 0.200 μg/L to 180 μg/L for all animals, with no abnormalities found on ECG and electrocardiography.

In blood clinical pathology, transient increases in AST and ALT levels were found to be included in all animals on day 3, and resolved without intervention on days 7 to 14 (fig. 36A and 36B).

Total bilirubin (TBil) levels, platelet counts and White Blood Cell (WBC) counts were maintained within normal limits for all animals throughout the study (fig. 37A, 37B and 37C).

Coagulation data collected from animals throughout the study revealed a short rise in Prothrombin Time (PT), activated APTT and D-dimer levels in several animals on day 3 (fig. 38A, 38B and 38C). These transient increases subside without intervention.

For any peptide pool evaluated, no T cell response was observed by IFN- γelispot against human GLA transgene product or AAVhu68 capsid in PBMCs or lymphocytes from spleen, cardiac lymph nodes or liver from animals administered a single IV dose of AAVhu68.Hglaco (d2379c).

Histopathological examination of tissues collected at necropsy revealed that there was some slight (grade 1) inflammatory cell infiltration in some organs with a similar incidence and severity to typical background findings in historical controls and published literature on background findings. DRG and TRG neuronal degeneration and spinal dorsal axonal disease were absent or minimal.

Table: single intravenous administration of AAVhu68.HGLACO (D233 C_I 359C) (2.5x10) ¹³ Semi-quantitative histopathological scoring assessment of tissues collected from adult NHPs 60 days after GC/kg)

^a The numbers represent the semi-quantitative fractionation of the findings. 0 = within normal limits, 1 = minimum severity. The spinal cord and DRG were determined to be less than grade 1 (calculations are provided in brackets) by summing the number of grade 1 results observed and dividing by the total sections assessed for that tissue.

Neutralizing antibodies against the AAVhu68 capsid and non-neutralizing binding antibodies (babs) (i.e., immunoglobulin G [ IgG ] and immunoglobulin M [ IgM ]) did not reach detectable levels in any NHP at baseline, consistent with the screening of NAb-negative animals of this study (fig. 39). As expected, by day 60, all animals had detectable levels of NAb and IgG BAb against AAVhu68 capsids, while IgM BAb against AAVhu68 capsids remained below detectable limits.

In plasma, IV administration of aavhu68.Hglaco (d233 c_i 359C) resulted in significant levels of transgene product expression (GLA enzyme activity). Average GLA enzyme activity increased approximately 50-fold from day 0 to day 14 post-administration (fig. 40). GLA enzyme activity levels peaked on day 14 and then declined on day 60. At the final time point of the assessment (day 60), GLA enzyme activity was approximately 2-fold higher than baseline levels observed on day 0. This decrease in transgene product expression after day 14 was unexpected. Similar to previous NHP studies on AAV delivery of human transgene products, a decrease in transgene product expression was associated with a humoral immune response to foreign human transgene products (anti-human GLA antibodies) (fig. 41).

Intravenous administration of aavhu68.Hglaco (d233 c_i 359C) also resulted in significant levels of transgene product expression (GLA enzyme activity) in heart, liver and kidney 60 days post-treatment (fig. 42A). The greatest fold increase in GLA enzyme activity was observed in the heart and kidneys (fig. 42B). At 2.5x10 ¹³ The average fold increases in GLA enzyme activity in NHP were 4.4 (437%), 0.5 (51%) and 3.3 (325%) in heart, liver and kidney, respectively, at GC/kg. These are combined at 2.5x10 ¹² GC/kg and 5.0x10 ¹² The observed increases in KO mice at GC/kg dose were of similar magnitude (fig. 27-29), indicating strong GL-3 clearance (fig. 31A and 31B, 32A, 32B). Fig. 43 to 45 show transgene expression (ISH) and transgene product (IHC) in heart, kidney and DRG.

Cumulatively, the study confirmed that at 2.5x10 ¹³ The administration of aavhu68.Hglaco (d233 c_i 359C) at the dose of GC/kg was well tolerated in NHP and resulted in a significant increase in transgene expression (GLA enzyme activity) in the target tissues for the treatment of brile disease (kidney, heart, DRG).

Example 7: evaluation of high dose pharmacological study of cynomolgus monkey intravenous administration of AAVhu68.HGLACO (D233 C_1359C)

Adult NHP (n=3) received a single IV dose of aavhu68.Hglaco (d233 c_i 359C) at a dose of 5.0x10 ¹³ GC/kg. The in vivo evaluation included daily clinical observations, body weight, blood clinical pathology (CBC, coagulation panel, serum chemistry [ including cardiac biomarker troponin I ]]) And cardiac function assessment (ECG and echocardiography). All animals were necropsied at day 60±3 after vehicle administration. At necropsy, tissues were collected for histopathological examination. Target tissues were collected for vector biodistribution analysis and transgene product expression localization (human GLA ISH mRNA]And human GLAIHC [ protein ]]) Is a comprehensive histological evaluation of (c). PBMCs, splenocytes and hepatic lymphocytes were also collected to measure T cell responses to capsid and transgene products (IFN- γ ELIspot).

Abbreviations: ADA = anti-drug antibody; BL = baseline; ELISpot = enzyme linked immunosorbent assay; GLA = α -galactosidase a; IFN- γ = interferon γ; IHC = immunohistochemistry; IN = intranasal; ISH = in situ hybridization; mRNA = mRNA; ms=mass spectrum; nab = neutralizing antibody; PBMC = peripheral blood mononuclear cells.

^a BL was up to 21 days prior to dosing.

^b Serum chemistry analysis included troponin I (cardiac biomarker).

^c Samples were collected and stored for future analysis.

^d Tissue was collected for histopathology, vector biodistribution analysis, and transgene product expression (human GLA ISH staining [ mRNA ]And human GLA IHC staining [ protein]) Is a histological evaluation of (c). Liver and spleen lymphocytes were collected for measuring T cell responses to capsid or transgene products (IFN- γ ELISpot).

Example 8: efficacy after IV administration of aavhu68.Hglaco (d233c_i 359C) to fabry disease mice to determine MED

The pharmacological study was aimed at determining the MED administered by aavhu68.Hglaco (d233 c_i 359C) IV in the severe fabry disease mouse model (Gla KO/TgG 3S) and assessing pharmacology and histopathology (efficacy and safety). The study included n=144 animals and two necropsy time points. Four dose levels of the vehicle were assessed. Dose levels were selected based on experimental efficacy data in mice and NHPs.

As summarized in the following table, at one of the four dose levels to be determined (5.0x10 ¹² GC/kg、1.0x10 ¹³ GC/kg、2.5x10 ¹³ GC/kg or 5.0x10 ¹³ GC/kg) or vehicle (PBS) to adult (2 to 3 months old) male-aggravated Fabry-Perot mice (Gla KO/TgG 3S) were administered AAVhu68.HGLaco (D233 C_I 359C). Normal male WT and WT/TgG3S males have been administered vehicle as controls. Female-weighted Gla KO/TgG3S mice also received the highest dose (5.0x10 ¹³ GC/kg) AAVhu68.HGLaco (D233C_1359C) or vehicle. Each group included 16 animals.Half of the animals (8 per group) will be sacrificed on study day 120 and the other half (8 per group) will undergo necropsy when at least 80% of the vehicle-treated Gla KO/TgG3S mice have reached the humane endpoint (defined as severe tremors and ataxia leading to walking disorders and/or body weight loss of ≡20% peak body weight).

And (3) shrinkage and: f = female; gla = α -galactosidase a (gene); IV = intravenous; KO = knockout; m = male; n/a = inapplicable; ROA = route of administration; tbd=to be determined; WT = wild type.

The in vivo evaluation included daily viability checks to monitor survival, weight measurements, clinical observations, thermo-sensory function evaluation (hotplate latency), serum urea nitrogen (BUN) levels, urine osmotic pressure, urine volume, and serum transgene expression (GLA enzyme activity) evaluations. Necropsy was performed at the humane end-point and at an earlier time point (day 120). At necropsy, a complete tissue list was collected for histopathological evaluation. Additional tissues (DRG, heart, kidney) were collected to assess GL-3 storage (GL-3 IHC). Target tissues were also collected for transgene expression assays (GLA enzyme activity) and quantification of GL-3 (brain, heart, kidney, liver, large intestine) by LC-MS/MS. Blood was collected for CBC/classification and serum clinical chemistry analysis, including BUN levels. Plasma was also collected to evaluate transgene product expression (GLA enzyme activity) and lyso-Gb ₃ And (5) storing.

The treatment conditions and genotypes of each mouse were unknown to the person performing the living body weight evaluation and the hot plate test.

MED is determined based on survival benefits, body weight, thermo-sensory function (assessed using a hotplate assay), renal function (assessed by BUN levels, urine volume and urine osmolality), correction of GL-3 lysosomal storage in target tissues, and analysis of transgene product expression (GAL activity levels) in disease-associated target organs.

Example 9: toxicology study of intravenous administration of AAVhu68.HGLACO (D233 C_I 359C) to adult non-human primate

A 180 day GLP compliance toxicology study was performed to evaluate aavhu68.Hglaco (d233 c_i359C) at low dose (1.0x10 ¹³ GC/kg), medium dose (2.5x10 ¹³ GC/kg) or high dose (5.0x10 ¹³ GC/kg) safety, tolerability, transgene product expression, biodistribution and excretion status after single IV administration to cynomolgus NHP. Additional NHPs were administered vehicle (PBS) as a control.

NHP (cynomolgus monkey) was selected for planned toxicology studies. This model was chosen because we have a lot of experience with the use of AAV vectors in NHPs, and the toxicology and immune response of NHPs closely represent that of humans. Adult NHP (2 to 8 years) was selected to represent the patient population for which clinical trials were planned. Male and female will be included in the study.

The IV route was chosen because systemic administration provided optimal transduction and transgene product expression in disease-related target tissues (DRG, kidney and heart) and non-disease-related (liver) target tissues.

Table-NHP toxicology study group

Cage-side clinical observations and assessments of vital signs, body weight, blood clinical pathology (CBC versus differential, clinical chemistry, coagulation panels) and CSF (cytology and chemistry) were made at frequent time intervals throughout the course of the study. Whole blood count (CBC), liver parameters and complement activation were monitored as acute hepatotoxicity, thrombocytopenia and complement activation are known toxicities following systemic AAV administration.

Troponin I test was included as part of the clinical pathology group along with baseline and echocardiographic evaluation every 30 days after treatment to monitor for signs of cardiac toxicity, as AAVhu68 showed high tropism to cardiac tissue after IV administration.

Neurological examinations were performed at baseline, day 14, day 28 and every 30 days thereafter. Advances at baseline, day 28, day 60, and day 180Sensory Nerve Conduction Studies (NCS) of bilateral median nerves were performed to monitor signs of DRG sensory neuron degeneration. These time points were selected based on the known kinetics of sensory neuron degeneration in NHP, which occurred 14 to 21 days after vector administration and were detectable on the median NCS by day 30. For neurological examination, the assessment was divided into five parts, assessing mental state, posture and gait, proprioception, cranial nerves and spinal reflex. The tests for each evaluation were performed in the same order. Neurological examination evaluators were blind to the treatment group; however, the evaluator is typically unaware of the treatment group at the time of evaluation. Where applicable, each assessment category was given a numerical score and recorded (unusual: 1; abnormal: 2; decline: 3; increase: 4; none: 5;N/A: unsuitable). For sensory NCS, nicolet was used System (Natus neurology Co.) and +.>Analysis software measures sensory nerve action potential amplitude and conduction velocity. The panelists who performed the NCS analysis officially set blinds to the treatment group.

Expression of the transgene product (GLA enzyme activity) was measured in serum. Samples were collected at frequent intervals during the expected onset, peak and plateau of transgene expression. Anti-transgene product antibodies (i.e., anti-drug antibodies [ ADA ]) were also evaluated in serum at corresponding time points using enzyme-linked immunosorbent assays (ELISA) to assess potential antibody responses to foreign human transgene products that may occur systemically.

Neutralizing antibody responses against AAVhu68 capsids were measured at baseline to assess the effect on vector transduction (biodistribution) and then the kinetics of NAb responses were assessed monthly. Peripheral blood mononuclear cells were collected to assess T cell responses to capsid and/or transgene products using an IFN- γ ELISpot assay. The time point for PBMC collection was chosen because T cell and B cell immune responses typically occurred in NHP within 30 days. At necropsy, tissue resident lymphocytes from spleen and liver were also collected for evaluation of T cell responses to capsid and/or transgene products.

Serum and CSF were collected to assess carrier distribution, urine and feces were collected to assess carrier excretion (shedding). These samples were collected at frequent time points and quantified by quantitative polymerase chain reaction (qPCR) to enable assessment of the kinetics of post-treatment carrier distribution and excretion. CSF and serum samples were also collected and archived for future analysis in case any findings require analysis.

At day 180 necropsy, a complete tissue list was collected for histopathology and carrier biodistribution analysis. Tissues were also collected to assess transgene product expression. All tissues were selected to include possible target tissues for fabry disease (kidneys, heart, DRG, intestines) and/or highly perfused peripheral organs (such as liver and kidneys). In addition, lymphocytes were harvested from liver, spleen and bone marrow to assess the presence of T cells in these organs that were reactive with capsid and/or transgene products at necropsy.

(sequence listing of independent text)

The following information is provided for a sequence containing independent text under the numeric identifier <223 >.

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All patent and non-patent publications cited in this specification are herein incorporated by reference in their entirety. International patent application number PCT/US2019/05567, filing date 2019, 10 months 10, U.S. provisional patent application number 63/089,850, filing date 2020, 10 months 9, U.S. provisional patent application number 63/146,286, filing date 2021, 2 months 5, U.S. provisional patent application number 63/186,092, filing date 2021, 5 months 8, the entire contents of which are incorporated herein by reference. The sequence listing filed herein under the name "19-8855pct_st25.Txt" and sequences and text therein are incorporated herein by reference. Although the invention has been described with reference to specific embodiments, it will be appreciated that modifications may be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

Sequence listing

<110> board of university of pennsylvania (The Trustees of the University of Pennsylvania)

<120> compositions and methods for treating brile disease

<130> UPN-19-8855.PCT

<150> US 63/089,850

<151> 2020-10-09

<150> US 63/146,286

<151> 2021-02-05

<150> US 63/186,092

<151> 2021-05-08

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<170> patent in version 3.5

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Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu

1 5 10 15

cgc ttc ctg gcc ctc gtt tcc tgg gac atc cct ggg gct aga gca ctg 96

Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu

20 25 30

gac aat gga ttg gca agg acg cct acc atg ggc tgg ctg cac tgg gag 144

Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu

35 40 45

cgc ttc atg tgc aac ctt gac tgc cag gaa gag cca gat tcc tgc atc 192

Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile

50 55 60

agt gag aag ctc ttc atg gag atg gca gag ctc atg gtc tca gaa ggc 240

Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly

65 70 75 80

tgg aag gat gca ggt tat gag tac ctc tgc att gat gac tgt tgg atg 288

Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met

85 90 95

gct ccc caa aga gat tca gaa ggc aga ctt cag gca gac cct cag cgc 336

Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg

100 105 110

ttt cct cat ggg att cgc cag cta gct aat tat gtt cac agc aaa gga 384

Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys Gly

115 120 125

ctg aag cta ggg att tat gca gat gtt gga aat aaa acc tgc gca ggc 432

Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly

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ttc cct ggg agt ttt gga tac tac gac att gat gcc cag acc ttt gct 480

Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala

145 150 155 160

gac tgg gga gta gat ctg cta aaa ttt gat ggt tgt tac tgt gac agt 528

Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser

165 170 175

ttg gaa aat ttg gca gat ggt tat aag cac atg tcc ttg gcc ctg aat 576

Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn

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Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met

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Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn

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His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys

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Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val

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Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn

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Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala

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Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser

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Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn

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Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn

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Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala

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Met Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile Ala

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Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile

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Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr

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Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln

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Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu

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Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu

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Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu

20 25 30

Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu

35 40 45

Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile

50 55 60

Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly

65 70 75 80

Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met

85 90 95

Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg

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Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly

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Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser

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Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn

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Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala

275 280 285

Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser

290 295 300

Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn

305 310 315 320

Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn

325 330 335

Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala

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Met Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile Ala

355 360 365

Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile

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atgcagctga gaaatcccga gctgcacctg ggctgtgccc tggctctgag atttctggcc 60

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tggaaggacg ccggctacga gtacctgtgc atcgacgact gttggatggc ccctcagaga 300

gactctgagg gcagactgca ggccgatcct cagagatttc cccacggcat tagacagctg 360

gccaactacg tgcacagcaa gggcctgaag ctgggcatct acgccgacgt gggcaacaag 420

acctgtgccg gctttcctgg cagcttcggc tactacgata tcgacgccca gaccttcgcc 480

gattggggag tcgatctgct gaagttcgac ggctgctact gcgacagcct ggaaaatctg 540

gccgacggct acaagcacat gtcactggcc ctgaatcgga ccggccgcag catcgtgtac 600

tcttgcgagt ggcccctgta tatgtggccc ttccagaagc ctaactacac cgagatcaga 660

cagtactgca accactggcg gaacttcgcc gacatcgacg atagctggaa gtccatcaag 720

agcatcctgg actggaccag cttcaatcaa gagcggatcg tggacgtggc aggacctggc 780

ggatggaacg atcctgacat gctggtcatc ggcaacttcg gcctgagctg gaaccagcaa 840

gtgacccaga tggccctgtg ggccattatg gccgctcctc tgttcatgag caacgacctg 900

agacacatca gccctcaggc caaggctctg ctgcaggaca aggatgtgat cgctatcaac 960

caggatcctc tgggcaagca gggctaccag ctgagacagg gcgacaattt cgaagtgtgg 1020

gaaagacccc tgagcggact ggcttgggcc gtcgccatga tcaacagaca agagatcggc 1080

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gcctgcttta tcacacagct gctgcccgtg aagagaaagc tgggctttta cgagtggacc 1200

agcagactgc ggagccacat caatcctacc ggcacagtgc tgctgcagct ggaaaacaca 1260

atgcagatga gcctgaagga cctgctg 1287

<210> 4

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<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

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atgcaactga gaaatcctga actgcacctg ggctgcgccc tggctctgag atttctggct 60

ctggtgtcct gggacatccc tggcgctaga gccctggata acggcctggc cagaacacct 120

acaatgggct ggctgcactg ggagagattc atgtgcaacc tggactgcca agaggaaccc 180

gacagctgca tcagcgagaa gctgttcatg gaaatggccg agctgatggt gtccgaaggc 240

tggaaggacg ccggctacga gtacctgtgc atcgacgact gttggatggc ccctcagaga 300

gactctgagg gcagactgca ggccgatcct cagagatttc cccacggcat tagacagctg 360

gccaactacg tgcacagcaa gggcctgaag ctgggcatct acgccgacgt gggcaacaag 420

acctgtgccg gctttcctgg cagcttcggc tactacgata tcgacgccca gaccttcgcc 480

gattggggag tcgatctgct gaagttcgac ggctgctact gcgacagcct ggaaaatctg 540

gccgacggct acaagcacat gtctctggcc ctgaatcgga ccggcagatc catcgtgtac 600

agctgcgagt ggcccctgta catgtggccc ttccagaagc ctaactacac cgagatcaga 660

cagtactgca accactggcg gaacttcgcc gacatctgcg atagctggaa gtccatcaag 720

agcatcctgg actggaccag cttcaatcaa gagcggatcg tggacgtggc aggacctggc 780

ggatggaacg atcctgacat gctggtcatc ggcaacttcg gcctgagctg gaaccagcaa 840

gtgacccaga tggccctgtg ggccattatg gccgctcctc tgttcatgag caacgacctg 900

agacacatca gccctcaggc caaggctctg ctgcaggaca aggatgtgat cgctatcaac 960

caggatcctc tgggcaagca gggctaccag ctgagacagg gcgacaattt cgaagtgtgg 1020

gaaagacccc tgagcggact ggcttgggcc gtcgccatga tcaaccggca agagtgcggc 1080

ggccccagat cctacacaat cgccgtggcc agtctcggca aaggcgtggc atgtaatccc 1140

gcctgcttca tcacacagct gctgcccgtg aagagaaagc tgggctttta cgagtggacc 1200

agcagactgc ggagccacat caatcctacc ggcacagtgc tgctgcagct ggaaaacacc 1260

atgcagatga gcctgaagga cctgctg 1287

<210> 5

<211> 1287

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

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atgcaactga gaaatcctga actgcacctg ggctgcgccc tggctctgag atttctggct 60

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acaatgggct ggctgcactg ggagagattc tgctgcaacc tggactgcca agaggaaccc 180

gacagctgca tcagcgagaa gctgttcatg gaaatggccg agctgatggt gtccgaaggc 240

tggaaggacg ccggctacga gtacctgtgc atcgacgact gttggatggc ccctcagaga 300

gactctgagg gcagactgca ggccgatcct cagagatttc cccacggcat tagacagctg 360

gccaactacg tgcacagcaa gggcctgaag ctgggcatct acgccgacgt gggcaacaag 420

acctgtgccg gctttcctgg cagcttcggc tactacgata tcgacgccca gaccttcgcc 480

gattggggag tcgatctgct gaagttcgac ggctgctact gcgacagcct ggaaaatctg 540

gccgacggct acaagcacat gtctctggcc ctgaatcgga ccggcagatc catcgtgtac 600

agctgcgagt ggcccctgta catgtggccc ttccagaagc ctaactacac cgagatcaga 660

cagtactgca accactggcg gaacttcgcc gacatcgacg atagctggaa gtccatcaag 720

agcatcctgg actggaccag cttcaatcaa gagcggatcg tggacgtggc aggacctggc 780

ggatggaacg atcctgacat gctggtcatc ggcaacttcg gcctgagctg gaaccagcaa 840

gtgacccaga tggccctgtg ggccattatg gccgctcctc tgttcatgag caacgacctg 900

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caggatcctc tgggcaagca gggctaccag ctgagacagg gcgacaattt cgaagtgtgg 1020

gaaagacccc tgagcggact ggcttgggcc gtcgccatga tcaaccggca agagatttgc 1080

ggccccagat cctacacaat cgccgtggcc agtctcggca aaggcgtggc atgtaatccc 1140

gcctgcttca tcacacagct gctgcccgtg aagagaaagc tgggctttta cgagtggacc 1200

agcagactgc ggagccacat caatcctacc ggcacagtgc tgctgcagct ggaaaacacc 1260

atgcagatga gcctgaagga cctgctg 1287

<210> 6

<211> 4297

<212> DNA

<213> artificial sequence

<220>

<223> CB7.CI.hGLAco(D233C/I359C).WPRE.rBG

<220>

<221> repeat_region

<222> (1)..(130)

<223> 5' ITR

<220>

<221> misc_feature

<222> (198)..(579)

<223> CMV immediate early enhancer

<220>

<221> promoter

<222> (582)..(863)

<223> CB promoter

<220>

<221> Intron

<222> (956)..(1928)

<223> chicken beta-actin intron

<220>

<221> CDS

<222> (1948)..(3240)

<223> hGLAco.D233C.I359C

<220>

<221> misc_feature

<222> (2353)..(2372)

<223> P18

<220>

<221> misc_feature

<222> (2644)..(2646)

<223> D233C

<220>

<221> misc_feature

<222> (3022)..(3024)

<223> I359C

<220>

<221> misc_feature

<222> (3253)..(3841)

<223> WPRE

<220>

<221> misc_feature

<222> (3609)..(3628)

<223> P19

<220>

<221> polyA_signal

<222> (3953)..(4079)

<223> Rabbit globin poly A

<220>

<221> repeat_region

<222> (4168)..(4297)

<223> 3' ITR

<400> 6

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctaccag ggtaatgggg 180

atcctctaga actatagcta gtcgacattg attattgact agttattaat agtaatcaat 240

tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 300

tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 360

tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 420

aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 480

caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 540

tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac 600

gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat 660

tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg 720

ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca 780

gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa 840

aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc 900

cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag 960

cgggcgggac ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt 1020

ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg 1080

gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc 1140

gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt 1200

gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa 1260

caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt 1320

cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg 1380

tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca 1440

ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg 1500

cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt 1560

atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa 1620

atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg 1680

caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc 1740

tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc 1800

ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 1860

cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt 1920

tggcaaagaa ttcgcggccg cgccacc atg caa ctg aga aat cct gaa ctg cac 1974

Met Gln Leu Arg Asn Pro Glu Leu His

1 5

ctg ggc tgc gcc ctg gct ctg aga ttt ctg gct ctg gtg tcc tgg gac 2022

Leu Gly Cys Ala Leu Ala Leu Arg Phe Leu Ala Leu Val Ser Trp Asp

10 15 20 25

atc cct ggc gct aga gcc ctg gat aac ggc ctg gcc aga aca cct aca 2070

Ile Pro Gly Ala Arg Ala Leu Asp Asn Gly Leu Ala Arg Thr Pro Thr

30 35 40

atg ggc tgg ctg cac tgg gag aga ttc atg tgc aac ctg gac tgc caa 2118

Met Gly Trp Leu His Trp Glu Arg Phe Met Cys Asn Leu Asp Cys Gln

45 50 55

gag gaa ccc gac agc tgc atc agc gag aag ctg ttc atg gaa atg gcc 2166

Glu Glu Pro Asp Ser Cys Ile Ser Glu Lys Leu Phe Met Glu Met Ala

60 65 70

gag ctg atg gtg tcc gaa ggc tgg aag gac gcc ggc tac gag tac ctg 2214

Glu Leu Met Val Ser Glu Gly Trp Lys Asp Ala Gly Tyr Glu Tyr Leu

75 80 85

tgc atc gac gac tgt tgg atg gcc cct cag aga gac tct gag ggc aga 2262

Cys Ile Asp Asp Cys Trp Met Ala Pro Gln Arg Asp Ser Glu Gly Arg

90 95 100 105

ctg cag gcc gat cct cag aga ttt ccc cac ggc att aga cag ctg gcc 2310

Leu Gln Ala Asp Pro Gln Arg Phe Pro His Gly Ile Arg Gln Leu Ala

110 115 120

aac tac gtg cac agc aag ggc ctg aag ctg ggc atc tac gcc gac gtg 2358

Asn Tyr Val His Ser Lys Gly Leu Lys Leu Gly Ile Tyr Ala Asp Val

125 130 135

ggc aac aag acc tgt gcc ggc ttt cct ggc agc ttc ggc tac tac gat 2406

Gly Asn Lys Thr Cys Ala Gly Phe Pro Gly Ser Phe Gly Tyr Tyr Asp

140 145 150

atc gac gcc cag acc ttc gcc gat tgg gga gtc gat ctg ctg aag ttc 2454

Ile Asp Ala Gln Thr Phe Ala Asp Trp Gly Val Asp Leu Leu Lys Phe

155 160 165

gac ggc tgc tac tgc gac agc ctg gaa aat ctg gcc gac ggc tac aag 2502

Asp Gly Cys Tyr Cys Asp Ser Leu Glu Asn Leu Ala Asp Gly Tyr Lys

170 175 180 185

cac atg tct ctg gcc ctg aat cgg acc ggc aga tcc atc gtg tac agc 2550

His Met Ser Leu Ala Leu Asn Arg Thr Gly Arg Ser Ile Val Tyr Ser

190 195 200

tgc gag tgg ccc ctg tac atg tgg ccc ttc cag aag cct aac tac acc 2598

Cys Glu Trp Pro Leu Tyr Met Trp Pro Phe Gln Lys Pro Asn Tyr Thr

205 210 215

gag atc aga cag tac tgc aac cac tgg cgg aac ttc gcc gac atc tgc 2646

Glu Ile Arg Gln Tyr Cys Asn His Trp Arg Asn Phe Ala Asp Ile Cys

220 225 230

gat agc tgg aag tcc atc aag agc atc ctg gac tgg acc agc ttc aat 2694

Asp Ser Trp Lys Ser Ile Lys Ser Ile Leu Asp Trp Thr Ser Phe Asn

235 240 245

caa gag cgg atc gtg gac gtg gca gga cct ggc gga tgg aac gat cct 2742

Gln Glu Arg Ile Val Asp Val Ala Gly Pro Gly Gly Trp Asn Asp Pro

250 255 260 265

gac atg ctg gtc atc ggc aac ttc ggc ctg agc tgg aac cag caa gtg 2790

Asp Met Leu Val Ile Gly Asn Phe Gly Leu Ser Trp Asn Gln Gln Val

270 275 280

acc cag atg gcc ctg tgg gcc att atg gcc gct cct ctg ttc atg agc 2838

Thr Gln Met Ala Leu Trp Ala Ile Met Ala Ala Pro Leu Phe Met Ser

285 290 295

aac gac ctg aga cac atc agc cct cag gcc aag gct ctg ctg cag gac 2886

Asn Asp Leu Arg His Ile Ser Pro Gln Ala Lys Ala Leu Leu Gln Asp

300 305 310

aag gat gtg atc gct atc aac cag gat cct ctg ggc aag cag ggc tac 2934

Lys Asp Val Ile Ala Ile Asn Gln Asp Pro Leu Gly Lys Gln Gly Tyr

315 320 325

cag ctg aga cag ggc gac aat ttc gaa gtg tgg gaa aga ccc ctg agc 2982

Gln Leu Arg Gln Gly Asp Asn Phe Glu Val Trp Glu Arg Pro Leu Ser

330 335 340 345

gga ctg gct tgg gcc gtc gcc atg atc aac cgg caa gag tgc ggc ggc 3030

Gly Leu Ala Trp Ala Val Ala Met Ile Asn Arg Gln Glu Cys Gly Gly

350 355 360

ccc aga tcc tac aca atc gcc gtg gcc agt ctc ggc aaa ggc gtg gca 3078

Pro Arg Ser Tyr Thr Ile Ala Val Ala Ser Leu Gly Lys Gly Val Ala

365 370 375

tgt aat ccc gcc tgc ttc atc aca cag ctg ctg ccc gtg aag aga aag 3126

Cys Asn Pro Ala Cys Phe Ile Thr Gln Leu Leu Pro Val Lys Arg Lys

380 385 390

ctg ggc ttt tac gag tgg acc agc aga ctg cgg agc cac atc aat cct 3174

Leu Gly Phe Tyr Glu Trp Thr Ser Arg Leu Arg Ser His Ile Asn Pro

395 400 405

acc ggc aca gtg ctg ctg cag ctg gaa aac acc atg cag atg agc ctg 3222

Thr Gly Thr Val Leu Leu Gln Leu Glu Asn Thr Met Gln Met Ser Leu

410 415 420 425

aag gac ctg ctg tga tag aagcttatcg ataatcaacc tctggattac 3270

Lys Asp Leu Leu

aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga 3330

tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc 3390

tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa 3450

cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc 3510

acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc 3570

atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc 3630

gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg 3690

attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct 3750

tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg 3810

agtcggatct ccctttgggc cgcctccccg catcgatacc gtctcgaatc aagcggccgc 3870

aagcatgctg agctccagat ctggtacctc tagagtcgac ccgggcggcc tcgaggacgg 3930

ggtgaactac gcctgaggat ccgatctttt tccctctgcc aaaaattatg gggacatcat 3990

gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca ttgcaatagt 4050

gtgttggaat tttttgtgtc tctcactcgg aagcaattcg ttgatctgaa tttcgaccac 4110

ccataatacc cattaccctg gtagataagt agcatggcgg gttaatcatt aactacaagg 4170

aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg 4230

ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag 4290

cgcgcag 4297

<210> 7

<211> 429

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 7

Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu

1 5 10 15

Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu

20 25 30

Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu

35 40 45

Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile

50 55 60

Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly

65 70 75 80

Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met

85 90 95

Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg

100 105 110

Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys Gly

115 120 125

Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly

130 135 140

Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala

145 150 155 160

Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser

165 170 175

Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn

180 185 190

Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met

195 200 205

Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn

210 215 220

His Trp Arg Asn Phe Ala Asp Ile Cys Asp Ser Trp Lys Ser Ile Lys

225 230 235 240

Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val

245 250 255

Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn

260 265 270

Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala

275 280 285

Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser

290 295 300

Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn

305 310 315 320

Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn

325 330 335

Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala

340 345 350

Met Ile Asn Arg Gln Glu Cys Gly Gly Pro Arg Ser Tyr Thr Ile Ala

355 360 365

Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile

370 375 380

Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr

385 390 395 400

Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln

405 410 415

Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu

420 425

<210> 8

<211> 3390

<212> DNA

<213> artificial sequence

<220>

<223> TBG.PI.hGLAnat.WPRE.bGH

<220>

<221> repeat_region

<222> (1)..(168)

<223> 5' ITR

<220>

<221> enhancer

<222> (211)..(310)

<223> α mic/bik

<220>

<221> enhancer

<222> (317)..(416)

<223> α mic/bik

<220>

<221> misc_feature

<222> (431)..(907)

<223> TBG promoter

<220>

<221> Intron

<222> (939)..(1071)

<223> SV40 misc intron

<220>

<221> CDS

<222> (1100)..(2392)

<223> hGLA Natural

<220>

<221> misc_feature

<222> (2411)..(2952)

<223> WPRE

<220>

<221> polyA_signal

<222> (2959)..(3173)

<223> BGH pA

<220>

<221> repeat_region

<222> (3223)..(3390)

<223> 3' ITR

<400> 8

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180

aggaagatcg gaattcgccc ttaagctagc aggttaattt ttaaaaagca gtcaaaagtc 240

caagtggccc ttggcagcat ttactctctc tgtttgctct ggttaataat ctcaggagca 300

caaacattcc agatccaggt taatttttaa aaagcagtca aaagtccaag tggcccttgg 360

cagcatttac tctctctgtt tgctctggtt aataatctca ggagcacaaa cattccagat 420

ccggcgcgcc agggctggaa gctacctttg acatcatttc ctctgcgaat gcatgtataa 480

tttctacaga acctattaga aaggatcacc cagcctctgc ttttgtacaa ctttccctta 540

aaaaactgcc aattccactg ctgtttggcc caatagtgag aactttttcc tgctgcctct 600

tggtgctttt gcctatggcc cctattctgc ctgctgaaga cactcttgcc agcatggact 660

taaacccctc cagctctgac aatcctcttt ctcttttgtt ttacatgaag ggtctggcag 720

ccaaagcaat cactcaaagt tcaaacctta tcattttttg ctttgttcct cttggccttg 780

gttttgtaca tcagctttga aaataccatc ccagggttaa tgctggggtt aatttataac 840

taagagtgct ctagttttgc aatacaggac atgctataaa aatggaaaga tgttgctttc 900

tgagagactg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag gttacaagac 960

aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc ttgcgtttct 1020

gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca ggtgtccagg 1080

cggccgcgaa ttcgccacc atg cag ctg agg aac cca gaa cta cat ctg ggc 1132

Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly

1 5 10

tgc gcg ctt gcg ctt cgc ttc ctg gcc ctc gtt tcc tgg gac atc cct 1180

Cys Ala Leu Ala Leu Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro

15 20 25

ggg gct aga gca ctg gac aat gga ttg gca agg acg cct acc atg ggc 1228

Gly Ala Arg Ala Leu Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly

30 35 40

tgg ctg cac tgg gag cgc ttc atg tgc aac ctt gac tgc cag gaa gag 1276

Trp Leu His Trp Glu Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu

45 50 55

cca gat tcc tgc atc agt gag aag ctc ttc atg gag atg gca gag ctc 1324

Pro Asp Ser Cys Ile Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu

60 65 70 75

atg gtc tca gaa ggc tgg aag gat gca ggt tat gag tac ctc tgc att 1372

Met Val Ser Glu Gly Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile

80 85 90

gat gac tgt tgg atg gct ccc caa aga gat tca gaa ggc aga ctt cag 1420

Asp Asp Cys Trp Met Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln

95 100 105

gca gac cct cag cgc ttt cct cat ggg att cgc cag cta gct aat tat 1468

Ala Asp Pro Gln Arg Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr

110 115 120

gtt cac agc aaa gga ctg aag cta ggg att tat gca gat gtt gga aat 1516

Val His Ser Lys Gly Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn

125 130 135

aaa acc tgc gca ggc ttc cct ggg agt ttt gga tac tac gac att gat 1564

Lys Thr Cys Ala Gly Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp

140 145 150 155

gcc cag acc ttt gct gac tgg gga gta gat ctg cta aaa ttt gat ggt 1612

Ala Gln Thr Phe Ala Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly

160 165 170

tgt tac tgt gac agt ttg gaa aat ttg gca gat ggt tat aag cac atg 1660

Cys Tyr Cys Asp Ser Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met

175 180 185

tcc ttg gcc ctg aat agg act ggc aga agc att gtg tac tcc tgt gag 1708

Ser Leu Ala Leu Asn Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu

190 195 200

tgg cct ctt tat atg tgg ccc ttt caa aag ccc aat tat aca gaa atc 1756

Trp Pro Leu Tyr Met Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile

205 210 215

cga cag tac tgc aat cac tgg cga aat ttt gct gac att gat gat tcc 1804

Arg Gln Tyr Cys Asn His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser

220 225 230 235

tgg aaa agt ata aag agt atc ttg gac tgg aca tct ttt aac cag gag 1852

Trp Lys Ser Ile Lys Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu

240 245 250

aga att gtt gat gtt gct gga cca ggg ggt tgg aat gac cca gat atg 1900

Arg Ile Val Asp Val Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met

255 260 265

tta gtg att ggc aac ttt ggc ctc agc tgg aat cag caa gta act cag 1948

Leu Val Ile Gly Asn Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln

270 275 280

atg gcc ctc tgg gct atc atg gct gct cct tta ttc atg tct aat gac 1996

Met Ala Leu Trp Ala Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp

285 290 295

ctc cga cac atc agc cct caa gcc aaa gct ctc ctt cag gat aag gac 2044

Leu Arg His Ile Ser Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp

300 305 310 315

gta att gcc atc aat cag gac ccc ttg ggc aag caa ggg tac cag ctt 2092

Val Ile Ala Ile Asn Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu

320 325 330

aga cag gga gac aac ttt gaa gtg tgg gaa cga cct ctc tca ggc tta 2140

Arg Gln Gly Asp Asn Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu

335 340 345

gcc tgg gct gta gct atg ata aac cgg cag gag att ggt gga cct cgc 2188

Ala Trp Ala Val Ala Met Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg

350 355 360

tct tat acc atc gca gtt gct tcc ctg ggt aaa gga gtg gcc tgt aat 2236

Ser Tyr Thr Ile Ala Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn

365 370 375

cct gcc tgc ttc atc aca cag ctc ctc cct gtg aaa agg aag cta ggg 2284

Pro Ala Cys Phe Ile Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly

380 385 390 395

ttc tat gaa tgg act tca agg tta aga agt cac ata aat ccc aca ggc 2332

Phe Tyr Glu Trp Thr Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly

400 405 410

act gtt ttg ctt cag cta gaa aat aca atg cag atg tca tta aaa gac 2380

Thr Val Leu Leu Gln Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp

415 420 425

tta ctt taa tga tgtacaaagc ttggatccaa tcaacctctg gattacaaaa 2432

Leu Leu

tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg 2492

ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct 2552

tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg 2612

gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct 2672

gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg 2732

ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg 2792

tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc 2852

tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc 2912

gcggcctgct gccggctctg cggcctcttc cgcgtcttcg agatctgcct cgactgtgcc 2972

ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg 3032

tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag 3092

gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga 3152

caatagcagg catgctgggg actcgagtta agggcgaatt cccgataagg atcttcctag 3212

agcatggcta cgtagataag tagcatggcg ggttaatcat taactacaag gaacccctag 3272

tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa 3332

aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcag 3390

<210> 9

<211> 429

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 9

Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu

1 5 10 15

Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu

20 25 30

Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu

35 40 45

Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile

50 55 60

Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly

65 70 75 80

Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met

85 90 95

Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg

100 105 110

Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys Gly

115 120 125

Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly

130 135 140

Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala

145 150 155 160

Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser

165 170 175

Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn

180 185 190

Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met

195 200 205

Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn

210 215 220

His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys

225 230 235 240

Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val

245 250 255

Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn

260 265 270

Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala

275 280 285

Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser

290 295 300

Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn

305 310 315 320

Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn

325 330 335

Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala

340 345 350

Met Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile Ala

355 360 365

Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile

370 375 380

Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr

385 390 395 400

Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln

405 410 415

Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu

420 425

<210> 10

<211> 4294

<212> DNA

<213> artificial sequence

<220>

<223> CB7.CI.hGLAnat.WPRE.RBG

<220>

<221> repeat_region

<222> (1)..(130)

<223> 5' ITR

<220>

<221> misc_feature

<222> (198)..(579)

<223> CMV IE promoter

<220>

<221> promoter

<222> (582)..(863)

<223> CB promoter

<220>

<221> TATA_signal

<222> (836)..(839)

<223> TATA

<220>

<221> Intron

<222> (956)..(1928)

<223> chicken beta-actin intron

<220>

<221> CDS

<222> (1950)..(3242)

<223> hGLA Natural

<220>

<221> misc_feature

<222> (3317)..(3905)

<223> WPRE

<220>

<221> polyA_signal

<222> (3950)..(4076)

<223> Rabbit globin poly A

<220>

<221> repeat_region

<222> (4165)..(4294)

<223> 3' ITR

<400> 10

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctaccag ggtaatgggg 180

atcctctaga actatagcta gtcgacattg attattgact agttattaat agtaatcaat 240

tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 300

tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 360

tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 420

aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 480

caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 540

tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac 600

gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat 660

tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg 720

ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca 780

gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa 840

aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc 900

cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag 960

cgggcgggac ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt 1020

ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg 1080

gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc 1140

gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt 1200

gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa 1260

caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt 1320

cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg 1380

tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca 1440

ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg 1500

cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt 1560

atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa 1620

atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg 1680

caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc 1740

tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc 1800

ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 1860

cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt 1920

tggcaaagaa tagcttcgaa ttcgccacc atg cag ctg agg aac cca gaa cta 1973

Met Gln Leu Arg Asn Pro Glu Leu

1 5

cat ctg ggc tgc gcg ctt gcg ctt cgc ttc ctg gcc ctc gtt tcc tgg 2021

His Leu Gly Cys Ala Leu Ala Leu Arg Phe Leu Ala Leu Val Ser Trp

10 15 20

gac atc cct ggg gct aga gca ctg gac aat gga ttg gca agg acg cct 2069

Asp Ile Pro Gly Ala Arg Ala Leu Asp Asn Gly Leu Ala Arg Thr Pro

25 30 35 40

acc atg ggc tgg ctg cac tgg gag cgc ttc atg tgc aac ctt gac tgc 2117

Thr Met Gly Trp Leu His Trp Glu Arg Phe Met Cys Asn Leu Asp Cys

45 50 55

cag gaa gag cca gat tcc tgc atc agt gag aag ctc ttc atg gag atg 2165

Gln Glu Glu Pro Asp Ser Cys Ile Ser Glu Lys Leu Phe Met Glu Met

60 65 70

gca gag ctc atg gtc tca gaa ggc tgg aag gat gca ggt tat gag tac 2213

Ala Glu Leu Met Val Ser Glu Gly Trp Lys Asp Ala Gly Tyr Glu Tyr

75 80 85

ctc tgc att gat gac tgt tgg atg gct ccc caa aga gat tca gaa ggc 2261

Leu Cys Ile Asp Asp Cys Trp Met Ala Pro Gln Arg Asp Ser Glu Gly

90 95 100

aga ctt cag gca gac cct cag cgc ttt cct cat ggg att cgc cag cta 2309

Arg Leu Gln Ala Asp Pro Gln Arg Phe Pro His Gly Ile Arg Gln Leu

105 110 115 120

gct aat tat gtt cac agc aaa gga ctg aag cta ggg att tat gca gat 2357

Ala Asn Tyr Val His Ser Lys Gly Leu Lys Leu Gly Ile Tyr Ala Asp

125 130 135

gtt gga aat aaa acc tgc gca ggc ttc cct ggg agt ttt gga tac tac 2405

Val Gly Asn Lys Thr Cys Ala Gly Phe Pro Gly Ser Phe Gly Tyr Tyr

140 145 150

gac att gat gcc cag acc ttt gct gac tgg gga gta gat ctg cta aaa 2453

Asp Ile Asp Ala Gln Thr Phe Ala Asp Trp Gly Val Asp Leu Leu Lys

155 160 165

ttt gat ggt tgt tac tgt gac agt ttg gaa aat ttg gca gat ggt tat 2501

Phe Asp Gly Cys Tyr Cys Asp Ser Leu Glu Asn Leu Ala Asp Gly Tyr

170 175 180

aag cac atg tcc ttg gcc ctg aat agg act ggc aga agc att gtg tac 2549

Lys His Met Ser Leu Ala Leu Asn Arg Thr Gly Arg Ser Ile Val Tyr

185 190 195 200

tcc tgt gag tgg cct ctt tat atg tgg ccc ttt caa aag ccc aat tat 2597

Ser Cys Glu Trp Pro Leu Tyr Met Trp Pro Phe Gln Lys Pro Asn Tyr

205 210 215

aca gaa atc cga cag tac tgc aat cac tgg cga aat ttt gct gac att 2645

Thr Glu Ile Arg Gln Tyr Cys Asn His Trp Arg Asn Phe Ala Asp Ile

220 225 230

gat gat tcc tgg aaa agt ata aag agt atc ttg gac tgg aca tct ttt 2693

Asp Asp Ser Trp Lys Ser Ile Lys Ser Ile Leu Asp Trp Thr Ser Phe

235 240 245

aac cag gag aga att gtt gat gtt gct gga cca ggg ggt tgg aat gac 2741

Asn Gln Glu Arg Ile Val Asp Val Ala Gly Pro Gly Gly Trp Asn Asp

250 255 260

cca gat atg tta gtg att ggc aac ttt ggc ctc agc tgg aat cag caa 2789

Pro Asp Met Leu Val Ile Gly Asn Phe Gly Leu Ser Trp Asn Gln Gln

265 270 275 280

gta act cag atg gcc ctc tgg gct atc atg gct gct cct tta ttc atg 2837

Val Thr Gln Met Ala Leu Trp Ala Ile Met Ala Ala Pro Leu Phe Met

285 290 295

tct aat gac ctc cga cac atc agc cct caa gcc aaa gct ctc ctt cag 2885

Ser Asn Asp Leu Arg His Ile Ser Pro Gln Ala Lys Ala Leu Leu Gln

300 305 310

gat aag gac gta att gcc atc aat cag gac ccc ttg ggc aag caa ggg 2933

Asp Lys Asp Val Ile Ala Ile Asn Gln Asp Pro Leu Gly Lys Gln Gly

315 320 325

tac cag ctt aga cag gga gac aac ttt gaa gtg tgg gaa cga cct ctc 2981

Tyr Gln Leu Arg Gln Gly Asp Asn Phe Glu Val Trp Glu Arg Pro Leu

330 335 340

tca ggc tta gcc tgg gct gta gct atg ata aac cgg cag gag att ggt 3029

Ser Gly Leu Ala Trp Ala Val Ala Met Ile Asn Arg Gln Glu Ile Gly

345 350 355 360

gga cct cgc tct tat acc atc gca gtt gct tcc ctg ggt aaa gga gtg 3077

Gly Pro Arg Ser Tyr Thr Ile Ala Val Ala Ser Leu Gly Lys Gly Val

365 370 375

gcc tgt aat cct gcc tgc ttc atc aca cag ctc ctc cct gtg aaa agg 3125

Ala Cys Asn Pro Ala Cys Phe Ile Thr Gln Leu Leu Pro Val Lys Arg

380 385 390

aag cta ggg ttc tat gaa tgg act tca agg tta aga agt cac ata aat 3173

Lys Leu Gly Phe Tyr Glu Trp Thr Ser Arg Leu Arg Ser His Ile Asn

395 400 405

ccc aca ggc act gtt ttg ctt cag cta gaa aat aca atg cag atg tca 3221

Pro Thr Gly Thr Val Leu Leu Gln Leu Glu Asn Thr Met Gln Met Ser

410 415 420

tta aaa gac tta ctt taa tga tgtacaagta aagatctgcg gccgcgtggt 3272

Leu Lys Asp Leu Leu

425

acctctagag tcgacccggg cggcctcgaa tcaagcttat cgataatcaa cctctggatt 3332

acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg 3392

gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct 3452

cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc 3512

aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca 3572

ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac 3632

tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt 3692

ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct 3752

ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc 3812

cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga 3872

cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtctcgag gacggggtga 3932

actacgcctg aggatccgat ctttttccct ctgccaaaaa ttatggggac atcatgaagc 3992

cccttgagca tctgacttct ggctaataaa ggaaatttat tttcattgca atagtgtgtt 4052

ggaatttttt gtgtctctca ctcggaagca attcgttgat ctgaatttcg accacccata 4112

atacccatta ccctggtaga taagtagcat ggcgggttaa tcattaacta caaggaaccc 4172

ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga 4232

ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc 4292

ag 4294

<210> 11

<211> 429

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 11

Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu

1 5 10 15

Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu

20 25 30

Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu

35 40 45

Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile

50 55 60

Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly

65 70 75 80

Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met

85 90 95

Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg

100 105 110

Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys Gly

115 120 125

Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly

130 135 140

Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala

145 150 155 160

Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser

165 170 175

Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn

180 185 190

Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met

195 200 205

Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn

210 215 220

His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys

225 230 235 240

Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val

245 250 255

Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn

260 265 270

Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala

275 280 285

Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser

290 295 300

Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn

305 310 315 320

Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn

325 330 335

Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala

340 345 350

Met Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile Ala

355 360 365

Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile

370 375 380

Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr

385 390 395 400

Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln

405 410 415

Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu

420 425

<210> 12

<211> 3390

<212> DNA

<213> artificial sequence

<220>

<223> TBG.PI.hGLAco.WPRE.bGH

<220>

<221> repeat_region

<222> (1)..(168)

<223> 5' ITR

<220>

<221> enhancer

<222> (211)..(310)

<220>

<221> enhancer

<222> (317)..(416)

<220>

<221> Intron

<222> (939)..(1071)

<223> SV40 misc intron

<220>

<221> CDS

<222> (1100)..(2392)

<223> hGLAco

<220>

<221> misc_feature

<222> (2411)..(2952)

<223> WPRE

<220>

<221> polyA_signal

<222> (2959)..(3173)

<223> BGH pA

<220>

<221> repeat_region

<222> (3223)..(3390)

<223> 3' ITR

<400> 12

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180

aggaagatcg gaattcgccc ttaagctagc aggttaattt ttaaaaagca gtcaaaagtc 240

caagtggccc ttggcagcat ttactctctc tgtttgctct ggttaataat ctcaggagca 300

caaacattcc agatccaggt taatttttaa aaagcagtca aaagtccaag tggcccttgg 360

cagcatttac tctctctgtt tgctctggtt aataatctca ggagcacaaa cattccagat 420

ccggcgcgcc agggctggaa gctacctttg acatcatttc ctctgcgaat gcatgtataa 480

tttctacaga acctattaga aaggatcacc cagcctctgc ttttgtacaa ctttccctta 540

aaaaactgcc aattccactg ctgtttggcc caatagtgag aactttttcc tgctgcctct 600

tggtgctttt gcctatggcc cctattctgc ctgctgaaga cactcttgcc agcatggact 660

taaacccctc cagctctgac aatcctcttt ctcttttgtt ttacatgaag ggtctggcag 720

ccaaagcaat cactcaaagt tcaaacctta tcattttttg ctttgttcct cttggccttg 780

gttttgtaca tcagctttga aaataccatc ccagggttaa tgctggggtt aatttataac 840

taagagtgct ctagttttgc aatacaggac atgctataaa aatggaaaga tgttgctttc 900

tgagagactg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag gttacaagac 960

aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc ttgcgtttct 1020

gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca ggtgtccagg 1080

cggccgcgaa ttcgccacc atg cag ctg aga aat ccc gag ctg cac ctg ggc 1132

Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly

1 5 10

tgt gcc ctg gct ctg aga ttt ctg gcc ctg gtg tct tgg gac atc cct 1180

Cys Ala Leu Ala Leu Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro

15 20 25

ggc gct aga gcc ctg gat aac ggc ctg gcc aga aca cct aca atg ggc 1228

Gly Ala Arg Ala Leu Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly

30 35 40

tgg ctg cac tgg gag aga ttc atg tgc aac ctg gac tgc caa gag gaa 1276

Trp Leu His Trp Glu Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu

45 50 55

ccc gac agc tgc atc agc gag aag ctg ttc atg gaa atg gcc gag ctg 1324

Pro Asp Ser Cys Ile Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu

60 65 70 75

atg gtg tcc gaa ggc tgg aag gac gcc ggc tac gag tac ctg tgc atc 1372

Met Val Ser Glu Gly Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile

80 85 90

gac gac tgt tgg atg gcc cct cag aga gac tct gag ggc aga ctg cag 1420

Asp Asp Cys Trp Met Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln

95 100 105

gcc gat cct cag aga ttt ccc cac ggc att aga cag ctg gcc aac tac 1468

Ala Asp Pro Gln Arg Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr

110 115 120

gtg cac agc aag ggc ctg aag ctg ggc atc tac gcc gac gtg ggc aac 1516

Val His Ser Lys Gly Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn

125 130 135

aag acc tgt gcc ggc ttt cct ggc agc ttc ggc tac tac gat atc gac 1564

Lys Thr Cys Ala Gly Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp

140 145 150 155

gcc cag acc ttc gcc gat tgg gga gtc gat ctg ctg aag ttc gac ggc 1612

Ala Gln Thr Phe Ala Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly

160 165 170

tgc tac tgc gac agc ctg gaa aat ctg gcc gac ggc tac aag cac atg 1660

Cys Tyr Cys Asp Ser Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met

175 180 185

tca ctg gcc ctg aat cgg acc ggc cgc agc atc gtg tac tct tgc gag 1708

Ser Leu Ala Leu Asn Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu

190 195 200

tgg ccc ctg tat atg tgg ccc ttc cag aag cct aac tac acc gag atc 1756

Trp Pro Leu Tyr Met Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile

205 210 215

aga cag tac tgc aac cac tgg cgg aac ttc gcc gac atc gac gat agc 1804

Arg Gln Tyr Cys Asn His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser

220 225 230 235

tgg aag tcc atc aag agc atc ctg gac tgg acc agc ttc aat caa gag 1852

Trp Lys Ser Ile Lys Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu

240 245 250

cgg atc gtg gac gtg gca gga cct ggc gga tgg aac gat cct gac atg 1900

Arg Ile Val Asp Val Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met

255 260 265

ctg gtc atc ggc aac ttc ggc ctg agc tgg aac cag caa gtg acc cag 1948

Leu Val Ile Gly Asn Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln

270 275 280

atg gcc ctg tgg gcc att atg gcc gct cct ctg ttc atg agc aac gac 1996

Met Ala Leu Trp Ala Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp

285 290 295

ctg aga cac atc agc cct cag gcc aag gct ctg ctg cag gac aag gat 2044

Leu Arg His Ile Ser Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp

300 305 310 315

gtg atc gct atc aac cag gat cct ctg ggc aag cag ggc tac cag ctg 2092

Val Ile Ala Ile Asn Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu

320 325 330

aga cag ggc gac aat ttc gaa gtg tgg gaa aga ccc ctg agc gga ctg 2140

Arg Gln Gly Asp Asn Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu

335 340 345

gct tgg gcc gtc gcc atg atc aac aga caa gag atc ggc gga ccc cgg 2188

Ala Trp Ala Val Ala Met Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg

350 355 360

tcc tac aca att gcc gtg gct tct ctc ggc aaa ggc gtg gcc tgt aat 2236

Ser Tyr Thr Ile Ala Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn

365 370 375

ccc gcc tgc ttt atc aca cag ctg ctg ccc gtg aag aga aag ctg ggc 2284

Pro Ala Cys Phe Ile Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly

380 385 390 395

ttt tac gag tgg acc agc aga ctg cgg agc cac atc aat cct acc ggc 2332

Phe Tyr Glu Trp Thr Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly

400 405 410

aca gtg ctg ctg cag ctg gaa aac aca atg cag atg agc ctg aag gac 2380

Thr Val Leu Leu Gln Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp

415 420 425

ctg ctg tga tga tgtacaaagc ttggatccaa tcaacctctg gattacaaaa 2432

Leu Leu

tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg 2492

ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct 2552

tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg 2612

gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct 2672

gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg 2732

ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg 2792

tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc 2852

tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc 2912

gcggcctgct gccggctctg cggcctcttc cgcgtcttcg agatctgcct cgactgtgcc 2972

ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg 3032

tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag 3092

gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga 3152

caatagcagg catgctgggg actcgagtta agggcgaatt cccgataagg atcttcctag 3212

agcatggcta cgtagataag tagcatggcg ggttaatcat taactacaag gaacccctag 3272

tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa 3332

aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcag 3390

<210> 13

<211> 429

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 13

Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu

1 5 10 15

Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu

20 25 30

Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu

35 40 45

Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile

50 55 60

Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly

65 70 75 80

Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met

85 90 95

Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg

100 105 110

Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys Gly

115 120 125

Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly

130 135 140

Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala

145 150 155 160

Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser

165 170 175

Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn

180 185 190

Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met

195 200 205

Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn

210 215 220

His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys

225 230 235 240

Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val

245 250 255

Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn

260 265 270

Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala

275 280 285

Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser

290 295 300

Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn

305 310 315 320

Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn

325 330 335

Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala

340 345 350

Met Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile Ala

355 360 365

Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile

370 375 380

Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr

385 390 395 400

Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln

405 410 415

Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu

420 425

<210> 14

<211> 4294

<212> DNA

<213> artificial sequence

<220>

<223> CB7.CI.hGLAco.WPRE.RBG

<220>

<221> repeat_region

<222> (1)..(130)

<223> 5' ITR

<220>

<221> repeat_region

<222> (198)..(579)

<223> CMV IE promoter

<220>

<221> promoter

<222> (582)..(863)

<223> CB promoter

<220>

<221> TATA_signal

<222> (582)..(863)

<223> TATA

<220>

<221> Intron

<222> (956)..(1928)

<223> chicken beta-actin intron

<220>

<221> CDS

<222> (1950)..(3242)

<223> hGLAco

<220>

<221> misc_feature

<222> (3317)..(3905)

<223> WPRE

<220>

<221> polyA_signal

<222> (3950)..(4076)

<223> Rabbit globin poly A

<220>

<221> repeat_region

<222> (4165)..(4294)

<223> 3' ITR

<400> 14

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctaccag ggtaatgggg 180

atcctctaga actatagcta gtcgacattg attattgact agttattaat agtaatcaat 240

tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 300

tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 360

tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 420

aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 480

caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 540

tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac 600

gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat 660

tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg 720

ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca 780

gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa 840

aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc 900

cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag 960

cgggcgggac ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt 1020

ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg 1080

gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc 1140

gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt 1200

gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa 1260

caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt 1320

cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg 1380

tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca 1440

ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg 1500

cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt 1560

atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa 1620

atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg 1680

caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc 1740

tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc 1800

ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 1860

cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt 1920

tggcaaagaa tagcttcgaa ttcgccacc atg cag ctg aga aat ccc gag ctg 1973

Met Gln Leu Arg Asn Pro Glu Leu

1 5

cac ctg ggc tgt gcc ctg gct ctg aga ttt ctg gcc ctg gtg tct tgg 2021

His Leu Gly Cys Ala Leu Ala Leu Arg Phe Leu Ala Leu Val Ser Trp

10 15 20

gac atc cct ggc gct aga gcc ctg gat aac ggc ctg gcc aga aca cct 2069

Asp Ile Pro Gly Ala Arg Ala Leu Asp Asn Gly Leu Ala Arg Thr Pro

25 30 35 40

aca atg ggc tgg ctg cac tgg gag aga ttc atg tgc aac ctg gac tgc 2117

Thr Met Gly Trp Leu His Trp Glu Arg Phe Met Cys Asn Leu Asp Cys

45 50 55

caa gag gaa ccc gac agc tgc atc agc gag aag ctg ttc atg gaa atg 2165

Gln Glu Glu Pro Asp Ser Cys Ile Ser Glu Lys Leu Phe Met Glu Met

60 65 70

gcc gag ctg atg gtg tcc gaa ggc tgg aag gac gcc ggc tac gag tac 2213

Ala Glu Leu Met Val Ser Glu Gly Trp Lys Asp Ala Gly Tyr Glu Tyr

75 80 85

ctg tgc atc gac gac tgt tgg atg gcc cct cag aga gac tct gag ggc 2261

Leu Cys Ile Asp Asp Cys Trp Met Ala Pro Gln Arg Asp Ser Glu Gly

90 95 100

aga ctg cag gcc gat cct cag aga ttt ccc cac ggc att aga cag ctg 2309

Arg Leu Gln Ala Asp Pro Gln Arg Phe Pro His Gly Ile Arg Gln Leu

105 110 115 120

gcc aac tac gtg cac agc aag ggc ctg aag ctg ggc atc tac gcc gac 2357

Ala Asn Tyr Val His Ser Lys Gly Leu Lys Leu Gly Ile Tyr Ala Asp

125 130 135

gtg ggc aac aag acc tgt gcc ggc ttt cct ggc agc ttc ggc tac tac 2405

Val Gly Asn Lys Thr Cys Ala Gly Phe Pro Gly Ser Phe Gly Tyr Tyr

140 145 150

gat atc gac gcc cag acc ttc gcc gat tgg gga gtc gat ctg ctg aag 2453

Asp Ile Asp Ala Gln Thr Phe Ala Asp Trp Gly Val Asp Leu Leu Lys

155 160 165

ttc gac ggc tgc tac tgc gac agc ctg gaa aat ctg gcc gac ggc tac 2501

Phe Asp Gly Cys Tyr Cys Asp Ser Leu Glu Asn Leu Ala Asp Gly Tyr

170 175 180

aag cac atg tca ctg gcc ctg aat cgg acc ggc cgc agc atc gtg tac 2549

Lys His Met Ser Leu Ala Leu Asn Arg Thr Gly Arg Ser Ile Val Tyr

185 190 195 200

tct tgc gag tgg ccc ctg tat atg tgg ccc ttc cag aag cct aac tac 2597

Ser Cys Glu Trp Pro Leu Tyr Met Trp Pro Phe Gln Lys Pro Asn Tyr

205 210 215

acc gag atc aga cag tac tgc aac cac tgg cgg aac ttc gcc gac atc 2645

Thr Glu Ile Arg Gln Tyr Cys Asn His Trp Arg Asn Phe Ala Asp Ile

220 225 230

gac gat agc tgg aag tcc atc aag agc atc ctg gac tgg acc agc ttc 2693

Asp Asp Ser Trp Lys Ser Ile Lys Ser Ile Leu Asp Trp Thr Ser Phe

235 240 245

aat caa gag cgg atc gtg gac gtg gca gga cct ggc gga tgg aac gat 2741

Asn Gln Glu Arg Ile Val Asp Val Ala Gly Pro Gly Gly Trp Asn Asp

250 255 260

cct gac atg ctg gtc atc ggc aac ttc ggc ctg agc tgg aac cag caa 2789

Pro Asp Met Leu Val Ile Gly Asn Phe Gly Leu Ser Trp Asn Gln Gln

265 270 275 280

gtg acc cag atg gcc ctg tgg gcc att atg gcc gct cct ctg ttc atg 2837

Val Thr Gln Met Ala Leu Trp Ala Ile Met Ala Ala Pro Leu Phe Met

285 290 295

agc aac gac ctg aga cac atc agc cct cag gcc aag gct ctg ctg cag 2885

Ser Asn Asp Leu Arg His Ile Ser Pro Gln Ala Lys Ala Leu Leu Gln

300 305 310

gac aag gat gtg atc gct atc aac cag gat cct ctg ggc aag cag ggc 2933

Asp Lys Asp Val Ile Ala Ile Asn Gln Asp Pro Leu Gly Lys Gln Gly

315 320 325

tac cag ctg aga cag ggc gac aat ttc gaa gtg tgg gaa aga ccc ctg 2981

Tyr Gln Leu Arg Gln Gly Asp Asn Phe Glu Val Trp Glu Arg Pro Leu

330 335 340

agc gga ctg gct tgg gcc gtc gcc atg atc aac aga caa gag atc ggc 3029

Ser Gly Leu Ala Trp Ala Val Ala Met Ile Asn Arg Gln Glu Ile Gly

345 350 355 360

gga ccc cgg tcc tac aca att gcc gtg gct tct ctc ggc aaa ggc gtg 3077

Gly Pro Arg Ser Tyr Thr Ile Ala Val Ala Ser Leu Gly Lys Gly Val

365 370 375

gcc tgt aat ccc gcc tgc ttt atc aca cag ctg ctg ccc gtg aag aga 3125

Ala Cys Asn Pro Ala Cys Phe Ile Thr Gln Leu Leu Pro Val Lys Arg

380 385 390

aag ctg ggc ttt tac gag tgg acc agc aga ctg cgg agc cac atc aat 3173

Lys Leu Gly Phe Tyr Glu Trp Thr Ser Arg Leu Arg Ser His Ile Asn

395 400 405

cct acc ggc aca gtg ctg ctg cag ctg gaa aac aca atg cag atg agc 3221

Pro Thr Gly Thr Val Leu Leu Gln Leu Glu Asn Thr Met Gln Met Ser

410 415 420

ctg aag gac ctg ctg tga tga tgtacaagta aagatctgcg gccgcgtggt 3272

Leu Lys Asp Leu Leu

425

acctctagag tcgacccggg cggcctcgaa tcaagcttat cgataatcaa cctctggatt 3332

acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg 3392

gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct 3452

cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc 3512

aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca 3572

ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac 3632

tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt 3692

ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct 3752

ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc 3812

cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga 3872

cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtctcgag gacggggtga 3932

actacgcctg aggatccgat ctttttccct ctgccaaaaa ttatggggac atcatgaagc 3992

cccttgagca tctgacttct ggctaataaa ggaaatttat tttcattgca atagtgtgtt 4052

ggaatttttt gtgtctctca ctcggaagca attcgttgat ctgaatttcg accacccata 4112

atacccatta ccctggtaga taagtagcat ggcgggttaa tcattaacta caaggaaccc 4172

ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga 4232

ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc 4292

ag 4294

<210> 15

<211> 429

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 15

Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu

1 5 10 15

Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu

20 25 30

Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu

35 40 45

Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile

50 55 60

Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly

65 70 75 80

Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met

85 90 95

Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg

100 105 110

Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys Gly

115 120 125

Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly

130 135 140

Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala

145 150 155 160

Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser

165 170 175

Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn

180 185 190

Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met

195 200 205

Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn

210 215 220

His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys

225 230 235 240

Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val

245 250 255

Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn

260 265 270

Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala

275 280 285

Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser

290 295 300

Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn

305 310 315 320

Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn

325 330 335

Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala

340 345 350

Met Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile Ala

355 360 365

Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile

370 375 380

Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr

385 390 395 400

Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln

405 410 415

Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu

420 425

<210> 16

<211> 3378

<212> DNA

<213> artificial sequence

<220>

<223> TBG.PI.hGLAco(M51C_G360C).WPRE.bGH

<220>

<221> repeat_region

<222> (1)..(168)

<223> 5' ITR

<220>

<221> enhancer

<222> (211)..(310)

<223> α mic/bik

<220>

<221> enhancer

<222> (317)..(416)

<223> α mic/bik

<220>

<221> misc_feature

<222> (431)..(907)

<223> TBG promoter

<220>

<221> Intron

<222> (939)..(1071)

<223> SV40 misc intron

<220>

<221> CDS

<222> (1094)..(2386)

<223> hGLAco.M51C.G360C

<220>

<221> misc_feature

<222> (2399)..(2940)

<223> WPRE

<220>

<221> polyA_signal

<222> (2947)..(3161)

<223> BGH pA

<220>

<221> repeat_region

<222> (3211)..(3378)

<223> 3' ITR

<400> 16

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180

aggaagatcg gaattcgccc ttaagctagc aggttaattt ttaaaaagca gtcaaaagtc 240

caagtggccc ttggcagcat ttactctctc tgtttgctct ggttaataat ctcaggagca 300

caaacattcc agatccaggt taatttttaa aaagcagtca aaagtccaag tggcccttgg 360

cagcatttac tctctctgtt tgctctggtt aataatctca ggagcacaaa cattccagat 420

ccggcgcgcc agggctggaa gctacctttg acatcatttc ctctgcgaat gcatgtataa 480

tttctacaga acctattaga aaggatcacc cagcctctgc ttttgtacaa ctttccctta 540

aaaaactgcc aattccactg ctgtttggcc caatagtgag aactttttcc tgctgcctct 600

tggtgctttt gcctatggcc cctattctgc ctgctgaaga cactcttgcc agcatggact 660

taaacccctc cagctctgac aatcctcttt ctcttttgtt ttacatgaag ggtctggcag 720

ccaaagcaat cactcaaagt tcaaacctta tcattttttg ctttgttcct cttggccttg 780

gttttgtaca tcagctttga aaataccatc ccagggttaa tgctggggtt aatttataac 840

taagagtgct ctagttttgc aatacaggac atgctataaa aatggaaaga tgttgctttc 900

tgagagactg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag gttacaagac 960

aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc ttgcgtttct 1020

gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca ggtgtccagg 1080

cggccgcgcc acc atg caa ctg aga aat cct gaa ctg cac ctg ggc tgc 1129

Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys

1 5 10

gcc ctg gct ctg aga ttt ctg gct ctg gtg tcc tgg gac atc cct ggc 1177

Ala Leu Ala Leu Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly

15 20 25

gct aga gcc ctg gat aac ggc ctg gcc aga aca cct aca atg ggc tgg 1225

Ala Arg Ala Leu Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp

30 35 40

ctg cac tgg gag aga ttc tgc tgc aac ctg gac tgc caa gag gaa ccc 1273

Leu His Trp Glu Arg Phe Cys Cys Asn Leu Asp Cys Gln Glu Glu Pro

45 50 55 60

gac agc tgc atc agc gag aag ctg ttc atg gaa atg gcc gag ctg atg 1321

Asp Ser Cys Ile Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met

65 70 75

gtg tcc gaa ggc tgg aag gac gcc ggc tac gag tac ctg tgc atc gac 1369

Val Ser Glu Gly Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp

80 85 90

gac tgt tgg atg gcc cct cag aga gac tct gag ggc aga ctg cag gcc 1417

Asp Cys Trp Met Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala

95 100 105

gat cct cag aga ttt ccc cac ggc att aga cag ctg gcc aac tac gtg 1465

Asp Pro Gln Arg Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val

110 115 120

cac agc aag ggc ctg aag ctg ggc atc tac gcc gac gtg ggc aac aag 1513

His Ser Lys Gly Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys

125 130 135 140

acc tgt gcc ggc ttt cct ggc agc ttc ggc tac tac gat atc gac gcc 1561

Thr Cys Ala Gly Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala

145 150 155

cag acc ttc gcc gat tgg gga gtc gat ctg ctg aag ttc gac ggc tgc 1609

Gln Thr Phe Ala Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys

160 165 170

tac tgc gac agc ctg gaa aat ctg gcc gac ggc tac aag cac atg tct 1657

Tyr Cys Asp Ser Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser

175 180 185

ctg gcc ctg aat cgg acc ggc aga tcc atc gtg tac agc tgc gag tgg 1705

Leu Ala Leu Asn Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp

190 195 200

ccc ctg tac atg tgg ccc ttc cag aag cct aac tac acc gag atc aga 1753

Pro Leu Tyr Met Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg

205 210 215 220

cag tac tgc aac cac tgg cgg aac ttc gcc gac atc gac gat agc tgg 1801

Gln Tyr Cys Asn His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp

225 230 235

aag tcc atc aag agc atc ctg gac tgg acc agc ttc aat caa gag cgg 1849

Lys Ser Ile Lys Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg

240 245 250

atc gtg gac gtg gca gga cct ggc gga tgg aac gat cct gac atg ctg 1897

Ile Val Asp Val Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu

255 260 265

gtc atc ggc aac ttc ggc ctg agc tgg aac cag caa gtg acc cag atg 1945

Val Ile Gly Asn Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met

270 275 280

gcc ctg tgg gcc att atg gcc gct cct ctg ttc atg agc aac gac ctg 1993

Ala Leu Trp Ala Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu

285 290 295 300

aga cac atc agc cct cag gcc aag gct ctg ctg cag gac aag gat gtg 2041

Arg His Ile Ser Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val

305 310 315

atc gct atc aac cag gat cct ctg ggc aag cag ggc tac cag ctg aga 2089

Ile Ala Ile Asn Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg

320 325 330

cag ggc gac aat ttc gaa gtg tgg gaa aga ccc ctg agc gga ctg gct 2137

Gln Gly Asp Asn Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala

335 340 345

tgg gcc gtc gcc atg atc aac cgg caa gag att tgc ggc ccc aga tcc 2185

Trp Ala Val Ala Met Ile Asn Arg Gln Glu Ile Cys Gly Pro Arg Ser

350 355 360

tac aca atc gcc gtg gcc agt ctc ggc aaa ggc gtg gca tgt aat ccc 2233

Tyr Thr Ile Ala Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro

365 370 375 380

gcc tgc ttc atc aca cag ctg ctg ccc gtg aag aga aag ctg ggc ttt 2281

Ala Cys Phe Ile Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe

385 390 395

tac gag tgg acc agc aga ctg cgg agc cac atc aat cct acc ggc aca 2329

Tyr Glu Trp Thr Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr

400 405 410

gtg ctg ctg cag ctg gaa aac acc atg cag atg agc ctg aag gac ctg 2377

Val Leu Leu Gln Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu

415 420 425

ctg tga tag aagcttggat ccaatcaacc tctggattac aaaatttgtg 2426

Leu

aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt 2486

taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata 2546

aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg 2606

tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc 2666

tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct 2726

gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt 2786

cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg 2846

ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc 2906

tgctgccggc tctgcggcct cttccgcgtc ttcgagatct gcctcgactg tgccttctag 2966

ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac 3026

tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca 3086

ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag 3146

caggcatgct ggggactcga gttaagggcg aattcccgat aaggatcttc ctagagcatg 3206

gctacgtaga taagtagcat ggcgggttaa tcattaacta caaggaaccc ctagtgatgg 3266

agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg 3326

cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc ag 3378

<210> 17

<211> 429

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 17

Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu

1 5 10 15

Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu

20 25 30

Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu

35 40 45

Arg Phe Cys Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile

50 55 60

Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly

65 70 75 80

Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met

85 90 95

Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg

100 105 110

Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys Gly

115 120 125

Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly

130 135 140

Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala

145 150 155 160

Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser

165 170 175

Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn

180 185 190

Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met

195 200 205

Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn

210 215 220

His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys

225 230 235 240

Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val

245 250 255

Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn

260 265 270

Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala

275 280 285

Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser

290 295 300

Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn

305 310 315 320

Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn

325 330 335

Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala

340 345 350

Met Ile Asn Arg Gln Glu Ile Cys Gly Pro Arg Ser Tyr Thr Ile Ala

355 360 365

Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile

370 375 380

Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr

385 390 395 400

Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln

405 410 415

Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu

420 425

<210> 18

<211> 4240

<212> DNA

<213> artificial sequence

<220>

<223> CB7.CI.hGLAco(M51C_G360C).WPRE.RBG

<220>

<221> repeat_region

<222> (1)..(130)

<223> 5' ITR

<220>

<221> misc_feature

<222> (198)..(579)

<223> CMV IE promoter

<220>

<221> promoter

<222> (582)..(863)

<223> CB promoter

<220>

<221> TATA_signal

<222> (836)..(839)

<223> TATA

<220>

<221> Intron

<222> (956)..(1928)

<223> chicken beta-actin intron

<220>

<221> CDS

<222> (1958)..(3250)

<223> hGLAco.M51C.G360C

<220>

<221> misc_feature

<222> (2363)..(2382)

<223> P18

<220>

<221> misc_feature

<222> (3263)..(3851)

<223> WPRE

<220>

<221> misc_feature

<222> (3619)..(3638)

<223> P19

<220>

<221> polyA_signal

<222> (3896)..(4022)

<223> Rabbit globin poly A

<220>

<221> repeat_region

<222> (4111)..(4240)

<223> 3' ITR

<400> 18

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctaccag ggtaatgggg 180

atcctctaga actatagcta gtcgacattg attattgact agttattaat agtaatcaat 240

tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 300

tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 360

tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 420

aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 480

caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 540

tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac 600

gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat 660

tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg 720

ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca 780

gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa 840

aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc 900

cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag 960

cgggcgggac ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt 1020

ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg 1080

gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc 1140

gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt 1200

gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa 1260

caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt 1320

cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg 1380

tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca 1440

ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg 1500

cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt 1560

atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa 1620

atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg 1680

caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc 1740

tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc 1800

ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 1860

cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt 1920

tggcaaagaa tagcttcgaa ttcgcggccg cgccacc atg caa ctg aga aat cct 1975

Met Gln Leu Arg Asn Pro

1 5

gaa ctg cac ctg ggc tgc gcc ctg gct ctg aga ttt ctg gct ctg gtg 2023

Glu Leu His Leu Gly Cys Ala Leu Ala Leu Arg Phe Leu Ala Leu Val

10 15 20

tcc tgg gac atc cct ggc gct aga gcc ctg gat aac ggc ctg gcc aga 2071

Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu Asp Asn Gly Leu Ala Arg

25 30 35

aca cct aca atg ggc tgg ctg cac tgg gag aga ttc tgc tgc aac ctg 2119

Thr Pro Thr Met Gly Trp Leu His Trp Glu Arg Phe Cys Cys Asn Leu

40 45 50

gac tgc caa gag gaa ccc gac agc tgc atc agc gag aag ctg ttc atg 2167

Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile Ser Glu Lys Leu Phe Met

55 60 65 70

gaa atg gcc gag ctg atg gtg tcc gaa ggc tgg aag gac gcc ggc tac 2215

Glu Met Ala Glu Leu Met Val Ser Glu Gly Trp Lys Asp Ala Gly Tyr

75 80 85

gag tac ctg tgc atc gac gac tgt tgg atg gcc cct cag aga gac tct 2263

Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met Ala Pro Gln Arg Asp Ser

90 95 100

gag ggc aga ctg cag gcc gat cct cag aga ttt ccc cac ggc att aga 2311

Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg Phe Pro His Gly Ile Arg

105 110 115

cag ctg gcc aac tac gtg cac agc aag ggc ctg aag ctg ggc atc tac 2359

Gln Leu Ala Asn Tyr Val His Ser Lys Gly Leu Lys Leu Gly Ile Tyr

120 125 130

gcc gac gtg ggc aac aag acc tgt gcc ggc ttt cct ggc agc ttc ggc 2407

Ala Asp Val Gly Asn Lys Thr Cys Ala Gly Phe Pro Gly Ser Phe Gly

135 140 145 150

tac tac gat atc gac gcc cag acc ttc gcc gat tgg gga gtc gat ctg 2455

Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala Asp Trp Gly Val Asp Leu

155 160 165

ctg aag ttc gac ggc tgc tac tgc gac agc ctg gaa aat ctg gcc gac 2503

Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser Leu Glu Asn Leu Ala Asp

170 175 180

ggc tac aag cac atg tct ctg gcc ctg aat cgg acc ggc aga tcc atc 2551

Gly Tyr Lys His Met Ser Leu Ala Leu Asn Arg Thr Gly Arg Ser Ile

185 190 195

gtg tac agc tgc gag tgg ccc ctg tac atg tgg ccc ttc cag aag cct 2599

Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met Trp Pro Phe Gln Lys Pro

200 205 210

aac tac acc gag atc aga cag tac tgc aac cac tgg cgg aac ttc gcc 2647

Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn His Trp Arg Asn Phe Ala

215 220 225 230

gac atc gac gat agc tgg aag tcc atc aag agc atc ctg gac tgg acc 2695

Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys Ser Ile Leu Asp Trp Thr

235 240 245

agc ttc aat caa gag cgg atc gtg gac gtg gca gga cct ggc gga tgg 2743

Ser Phe Asn Gln Glu Arg Ile Val Asp Val Ala Gly Pro Gly Gly Trp

250 255 260

aac gat cct gac atg ctg gtc atc ggc aac ttc ggc ctg agc tgg aac 2791

Asn Asp Pro Asp Met Leu Val Ile Gly Asn Phe Gly Leu Ser Trp Asn

265 270 275

cag caa gtg acc cag atg gcc ctg tgg gcc att atg gcc gct cct ctg 2839

Gln Gln Val Thr Gln Met Ala Leu Trp Ala Ile Met Ala Ala Pro Leu

280 285 290

ttc atg agc aac gac ctg aga cac atc agc cct cag gcc aag gct ctg 2887

Phe Met Ser Asn Asp Leu Arg His Ile Ser Pro Gln Ala Lys Ala Leu

295 300 305 310

ctg cag gac aag gat gtg atc gct atc aac cag gat cct ctg ggc aag 2935

Leu Gln Asp Lys Asp Val Ile Ala Ile Asn Gln Asp Pro Leu Gly Lys

315 320 325

cag ggc tac cag ctg aga cag ggc gac aat ttc gaa gtg tgg gaa aga 2983

Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn Phe Glu Val Trp Glu Arg

330 335 340

ccc ctg agc gga ctg gct tgg gcc gtc gcc atg atc aac cgg caa gag 3031

Pro Leu Ser Gly Leu Ala Trp Ala Val Ala Met Ile Asn Arg Gln Glu

345 350 355

att tgc ggc ccc aga tcc tac aca atc gcc gtg gcc agt ctc ggc aaa 3079

Ile Cys Gly Pro Arg Ser Tyr Thr Ile Ala Val Ala Ser Leu Gly Lys

360 365 370

ggc gtg gca tgt aat ccc gcc tgc ttc atc aca cag ctg ctg ccc gtg 3127

Gly Val Ala Cys Asn Pro Ala Cys Phe Ile Thr Gln Leu Leu Pro Val

375 380 385 390

aag aga aag ctg ggc ttt tac gag tgg acc agc aga ctg cgg agc cac 3175

Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr Ser Arg Leu Arg Ser His

395 400 405

atc aat cct acc ggc aca gtg ctg ctg cag ctg gaa aac acc atg cag 3223

Ile Asn Pro Thr Gly Thr Val Leu Leu Gln Leu Glu Asn Thr Met Gln

410 415 420

atg agc ctg aag gac ctg ctg tga tag aagcttatcg ataatcaacc 3270

Met Ser Leu Lys Asp Leu Leu

425

tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac 3330

gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt 3390

cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt 3450

tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg 3510

cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac 3570

ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac 3630

tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt 3690

tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc 3750

ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg 3810

ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc gtctcgagga 3870

cggggtgaac tacgcctgag gatccgatct ttttccctct gccaaaaatt atggggacat 3930

catgaagccc cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat 3990

agtgtgttgg aattttttgt gtctctcact cggaagcaat tcgttgatct gaatttcgac 4050

cacccataat acccattacc ctggtagata agtagcatgg cgggttaatc attaactaca 4110

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 4170

ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 4230

gagcgcgcag 4240

<210> 19

<211> 429

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 19

Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu

1 5 10 15

Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu

20 25 30

Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu

35 40 45

Arg Phe Cys Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile

50 55 60

Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly

65 70 75 80

Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met

85 90 95

Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg

100 105 110

Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys Gly

115 120 125

Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly

130 135 140

Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala

145 150 155 160

Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser

165 170 175

Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn

180 185 190

Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met

195 200 205

Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn

210 215 220

His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys

225 230 235 240

Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val

245 250 255

Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn

260 265 270

Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala

275 280 285

Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser

290 295 300

Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn

305 310 315 320

Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn

325 330 335

Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala

340 345 350

Met Ile Asn Arg Gln Glu Ile Cys Gly Pro Arg Ser Tyr Thr Ile Ala

355 360 365

Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile

370 375 380

Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr

385 390 395 400

Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln

405 410 415

Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu

420 425

<210> 20

<211> 2211

<212> DNA

<213> adeno-associated Virus human 68

<220>

<221> CDS

<222> (1)..(2211)

<400> 20

atg gct gcc gat ggt tat ctt cca gat tgg ctc gag gac aac ctc agt 48

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

gaa ggc att cgc gag tgg tgg gct ttg aaa cct gga gcc cct caa ccc 96

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

aag gca aat caa caa cat caa gac aac gct cgg ggt ctt gtg ctt ccg 144

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

ggt tac aaa tac ctt gga ccc ggc aac gga ctc gac aag ggg gag ccg 192

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

gtc aac gaa gca gac gcg gcg gcc ctc gag cac gac aag gcc tac gac 240

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

cag cag ctc aag gcc gga gac aac ccg tac ctc aag tac aac cac gcc 288

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

gac gcc gag ttc cag gag cgg ctc aaa gaa gat acg tct ttt ggg ggc 336

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

aac ctc ggg cga gca gtc ttc cag gcc aaa aag agg ctt ctt gaa cct 384

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

ctt ggt ctg gtt gag gaa gcg gct aag acg gct cct gga aag aag agg 432

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

cct gta gag cag tct cct cag gaa ccg gac tcc tcc gtg ggt att ggc 480

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Val Gly Ile Gly

145 150 155 160

aaa tcg ggt gca cag ccc gct aaa aag aga ctc aat ttc ggt cag act 528

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

ggc gac aca gag tca gtc ccc gac cct caa cca atc gga gaa cct ccc 576

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

gca gcc ccc tca ggt gtg gga tct ctt aca atg gct tca ggt ggt ggc 624

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

gca cca gtg gca gac aat aac gaa ggt gcc gat gga gtg ggt agt tcc 672

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220

tcg gga aat tgg cat tgc gat tcc caa tgg ctg ggg gac aga gtc atc 720

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

acc acc agc acc cga acc tgg gcc ctg ccc acc tac aac aat cac ctc 768

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

tac aag caa atc tcc aac agc aca tct gga gga tct tca aat gac aac 816

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270

gcc tac ttc ggc tac agc acc ccc tgg ggg tat ttt gac ttc aac aga 864

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

ttc cac tgc cac ttc tca cca cgt gac tgg caa aga ctc atc aac aac 912

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

aac tgg gga ttc cgg cct aag cga ctc aac ttc aag ctc ttc aac att 960

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

cag gtc aaa gag gtt acg gac aac aat gga gtc aag acc atc gct aat 1008

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335

aac ctt acc agc acg gtc cag gtc ttc acg gac tca gac tat cag ctc 1056

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350

ccg tac gtg ctc ggg tcg gct cac gag ggc tgc ctc ccg ccg ttc cca 1104

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365

gcg gac gtt ttc atg att cct cag tac ggg tat cta acg ctt aat gat 1152

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380

gga agc caa gcc gtg ggt cgt tcg tcc ttt tac tgc ctg gaa tat ttc 1200

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

ccg tcg caa atg cta aga acg ggt aac aac ttc cag ttc agc tac gag 1248

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415

ttt gag aac gta cct ttc cat agc agc tat gct cac agc caa agc ctg 1296

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

gac cga ctc atg aat cca ctc atc gac caa tac ttg tac tat ctc tca 1344

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445

aag act att aac ggt tct gga cag aat caa caa acg cta aaa ttc agt 1392

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460

gtg gcc gga ccc agc aac atg gct gtc cag gga aga aac tac ata cct 1440

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480

gga ccc agc tac cga caa caa cgt gtc tca acc act gtg act caa aac 1488

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495

aac aac agc gaa ttt gct tgg cct gga gct tct tct tgg gct ctc aat 1536

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510

gga cgt aat agc ttg atg aat cct gga cct gct atg gcc agc cac aaa 1584

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

gaa gga gag gac cgt ttc ttt cct ttg tct gga tct tta att ttt ggc 1632

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540

aaa caa gga act gga aga gac aac gtg gat gcg gac aaa gtc atg ata 1680

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560

acc aac gaa gaa gaa att aaa act acc aac cca gta gca acg gag tcc 1728

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575

tat gga caa gtg gcc aca aac cac cag agt gcc caa gca cag gcg cag 1776

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln

580 585 590

acc ggc tgg gtt caa aac caa gga ata ctt ccg ggt atg gtt tgg cag 1824

Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln

595 600 605

gac aga gat gtg tac ctg caa gga ccc att tgg gcc aaa att cct cac 1872

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

acg gac ggc aac ttt cac cct tct ccg ctg atg gga ggg ttt gga atg 1920

Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met

625 630 635 640

aag cac ccg cct cct cag atc ctc atc aaa aac aca cct gta cct gcg 1968

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

gat cct cca acg gct ttc aac aag gac aag ctg aac tct ttc atc acc 2016

Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr

660 665 670

cag tat tct act ggc caa gtc agc gtg gag att gag tgg gag ctg cag 2064

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

aag gaa aac agc aag cgc tgg aac ccg gag atc cag tac act tcc aac 2112

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700

tat tac aag tct aat aat gtt gaa ttt gct gtt aat act gaa ggt gtt 2160

Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val

705 710 715 720

tat tct gaa ccc cgc ccc att ggc acc aga tac ctg act cgt aat ctg 2208

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

taa 2211

<210> 21

<211> 736

<212> PRT

<213> adeno-associated Virus human 68

<400> 21

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Val Gly Ile Gly

145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln

580 585 590

Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700

Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val

705 710 715 720

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 22

<211> 2208

<212> DNA

<213> adeno-associated Virus 9

<220>

<221> CDS

<222> (1)..(2208)

<223> AAV9 VP1 capsid

<400> 22

atg gct gcc gat ggt tat ctt cca gat tgg ctc gag gac aac ctt agt 48

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

gaa gga att cgc gag tgg tgg gct ttg aaa cct gga gcc cct caa ccc 96

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

aag gca aat caa caa cat caa gac aac gct cga ggt ctt gtg ctt ccg 144

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

ggt tac aaa tac ctt gga ccc ggc aac gga ctc gac aag ggg gag ccg 192

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

gtc aac gca gca gac gcg gcg gcc ctc gag cac gac aag gcc tac gac 240

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

cag cag ctc aag gcc gga gac aac ccg tac ctc aag tac aac cac gcc 288

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

gac gcc gag ttc cag gag cgg ctc aaa gaa gat acg tct ttt ggg ggc 336

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

aac ctc ggg cga gca gtc ttc cag gcc aaa aag agg ctt ctt gaa cct 384

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

ctt ggt ctg gtt gag gaa gcg gct aag acg gct cct gga aag aag agg 432

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

cct gta gag cag tct cct cag gaa ccg gac tcc tcc gcg ggt att ggc 480

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160

aaa tcg ggt gca cag ccc gct aaa aag aga ctc aat ttc ggt cag act 528

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

ggc gac aca gag tca gtc cca gac cct caa cca atc gga gaa cct ccc 576

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

gca gcc ccc tca ggt gtg gga tct ctt aca atg gct tca ggt ggt ggc 624

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

gca cca gtg gca gac aat aac gaa ggt gcc gat gga gtg ggt agt tcc 672

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220

tcg gga aat tgg cat tgc gat tcc caa tgg ctg ggg gac aga gtc atc 720

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

acc acc agc acc cga acc tgg gcc ctg ccc acc tac aac aat cac ctc 768

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

tac aag caa atc tcc aac agc aca tct gga gga tct tca aat gac aac 816

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270

gcc tac ttc ggc tac agc acc ccc tgg ggg tat ttt gac ttc aac aga 864

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

ttc cac tgc cac ttc tca cca cgt gac tgg cag cga ctc atc aac aac 912

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

aac tgg gga ttc cgg cct aag cga ctc aac ttc aag ctc ttc aac att 960

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

cag gtc aaa gag gtt acg gac aac aat gga gtc aag acc atc gcc aat 1008

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335

aac ctt acc agc acg gtc cag gtc ttc acg gac tca gac tat cag ctc 1056

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350

ccg tac gtg ctc ggg tcg gct cac gag ggc tgc ctc ccg ccg ttc cca 1104

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365

gcg gac gtt ttc atg att cct cag tac ggg tat ctg acg ctt aat gat 1152

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380

gga agc cag gcc gtg ggt cgt tcg tcc ttt tac tgc ctg gaa tat ttc 1200

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

ccg tcg caa atg cta aga acg ggt aac aac ttc cag ttc agc tac gag 1248

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415

ttt gag aac gta cct ttc cat agc agc tac gct cac agc caa agc ctg 1296

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

gac cga cta atg aat cca ctc atc gac caa tac ttg tac tat ctc tca 1344

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445

aag act att aac ggt tct gga cag aat caa caa acg cta aaa ttc agt 1392

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460

gtg gcc gga ccc agc aac atg gct gtc cag gga aga aac tac ata cct 1440

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480

gga ccc agc tac cga caa caa cgt gtc tca acc act gtg act caa aac 1488

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495

aac aac agc gaa ttt gct tgg cct gga gct tct tct tgg gct ctc aat 1536

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510

gga cgt aat agc ttg atg aat cct gga cct gct atg gcc agc cac aaa 1584

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

gaa gga gag gac cgt ttc ttt cct ttg tct gga tct tta att ttt ggc 1632

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540

aaa caa gga act gga aga gac aac gtg gat gcg gac aaa gtc atg ata 1680

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560

acc aac gaa gaa gaa att aaa act act aac ccg gta gca acg gag tcc 1728

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575

tat gga caa gtg gcc aca aac cac cag agt gcc caa gca cag gcg cag 1776

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln

580 585 590

acc ggc tgg gtt caa aac caa gga ata ctt ccg ggt atg gtt tgg cag 1824

Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln

595 600 605

gac aga gat gtg tac ctg caa gga ccc att tgg gcc aaa att cct cac 1872

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

acg gac ggc aac ttt cac cct tct ccg ctg atg gga ggg ttt gga atg 1920

Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met

625 630 635 640

aag cac ccg cct cct cag atc ctc atc aaa aac aca cct gta cct gcg 1968

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

gat cct cca acg gcc ttc aac aag gac aag ctg aac tct ttc atc acc 2016

Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr

660 665 670

cag tat tct act ggc caa gtc agc gtg gag atc gag tgg gag ctg cag 2064

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

aag gaa aac agc aag cgc tgg aac ccg gag atc cag tac act tcc aac 2112

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700

tat tac aag tct aat aat gtt gaa ttt gct gtt aat act gaa ggt gta 2160

Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val

705 710 715 720

tat agt gaa ccc cgc ccc att ggc acc aga tac ctg act cgt aat ctg 2208

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 23

<211> 736

<212> PRT

<213> adeno-associated Virus 9

<400> 23

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln

580 585 590

Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700

Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val

705 710 715 720

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 24

<211> 130

<212> DNA

<213> adeno-associated Virus 2

<400> 24

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct 130

<210> 25

<211> 130

<212> DNA

<213> adeno-associated Virus 2

<400> 25

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120

gagcgcgcag 130

<210> 26

<211> 973

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 26

gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg 60

cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc 120

ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc 180

tccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc 240

agtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag 300

gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg 360

tcggtcgggc tgcaaccccc cctgcacccc cctccccgag ttgctgagca cggcccggct 420

tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg ccgtgccggg cggggggtgg 480

cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg ccggggaggg ctcgggggag 540

gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg cggcgagccg cagccattgc 600

cttttatggt aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag 660

ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc gcggggcgaa gcggtgcggc 720

gccggcagga aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt 780

ctccctctcc agcctcgggg ctgtccgcgg ggggacggct gccttcgggg gggacggggc 840

agggcggggt tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt 900

catgccttct tctttttcct acagctcctg ggcaacgtgc tggttattgt gctgtctcat 960

cattttggca aag 973

<210> 27

<211> 589

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 27

aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 60

ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 120

atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 180

tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 240

ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct 300

attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 360

ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc 420

gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 480

aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt 540

cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgc 589

<210> 28

<211> 127

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 28

gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact 60

tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc 120

tcactcg 127

<210> 29

<211> 282

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 29

tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc tccccacccc 60

caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg gggggggggg 120

gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg aggcggagag 180

gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc 240

ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gg 282

<210> 30

<211> 382

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 30

ctagtcgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 60

atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 120

cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 180

tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag 240

tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 300

ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 360

acgtattagt catcgctatt ac 382

<210> 31

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> miR target sequences

<400> 31

agtgaattct accagtgcca ta 22

<210> 32

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> miR target sequences

<400> 32

agtgtgagtt ctaccattgc caaa 24

Claims

1. A recombinant AAV (rAAV) comprising an AAVhu68 capsid having packaged therein a vector genome, wherein said vector genome comprises a coding sequence for functional human alpha-galactosidase A (hGLA) and regulatory sequences that direct the expression of said hGLA in a target cell,

Wherein the coding sequence comprises SEQ ID NO:4, or a sequence at least 85% identical thereto, and wherein the hGLA is at a position based on SEQ ID NO:2 has a cysteine residue at position 233 and/or position 359.

2. The rAAV of claim 1, wherein the hGLA comprises SEQ ID NO:2, or a sequence at least 95% identical thereto.

3. The rAAV of claim 1 or 2, wherein the hGLA comprises SEQ ID NO:7 from amino acids 32 to 429.

4. The rAAV of any one of claims 1-3, wherein the hGLA comprises a native signal peptide.

5. The rAAV of any one of claims 1-3, wherein the hGLA comprises a heterologous signal peptide.

6. The rAAV of any one of claims 1-4, wherein the hGLA comprises SEQ ID NO:17 (amino acids 1 to 429), or a sequence at least 95% identical thereto.

7. The rAAV of any one of claims 1-6, wherein the vector genome comprises a tissue-specific promoter.

8. The rAAV of any one of claims 1-6, wherein the regulatory sequence comprises a CB7 promoter, an intron, and a polyA.

9. The rAAV of any one of claims 1-8, wherein the regulatory sequence comprises a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE).

10. The rAAV of claim 9, wherein the WPRE comprises SEQ ID NO:27.

11. the rAAV of any one of claims 1-9, wherein the vector genome comprises one or more miRNA target sequences.

12. The rAAV of claim 1, wherein the vector genome comprises a nucleotide sequence that hybridizes to SEQ ID NO:6, at least 85% identical sequence.

13. An expression cassette comprising a nucleic acid sequence encoding a functional human alpha-galactosidase A (hGLA) and one or more regulatory sequences directing the expression of said hGLA in a target cell comprising said expression cassette,

wherein the nucleic acid sequence comprises SEQ ID NO:4, or a sequence at least 85% identical thereto, and wherein the hGLA is at a position based on SEQ ID NO:2 or SEQ ID NO:7 has a cysteine residue at position 233 and/or position 359.

14. The expression cassette of claim 12, wherein the hGLA comprises SEQ ID NO:7 from amino acids 32 to 429.

15. The expression cassette of claim 12 or 13, wherein the hGLA comprises a natural signal peptide.

16. The expression cassette of any one of claims 12-14, wherein the hGLA comprises a heterologous signal peptide.

17. The expression cassette of any one of claims 12-15, wherein the hGLA comprises SEQ ID NO:7 (amino acids 1 to 429), or a sequence at least 95% identical thereto.

18. The expression cassette according to any one of claims 12 to 16, wherein the expression cassette comprises a tissue specific promoter.

19. The expression cassette of any one of claims 12 to 16, wherein the regulatory sequence comprises a CB7 promoter, an intron, and polyA.

20. The expression cassette of any one of claims 12-18, wherein the regulatory sequence comprises a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE).

21. The rAAV of claim 19, wherein the WPRE comprises SEQ ID NO:27.

22. the expression cassette of any one of claims 12 to 20, further comprising one or more miRNA target sequences.

23. The expression cassette according to any one of claims 12 to 21, wherein the expression cassette is carried by a non-viral vector or a viral vector.

24. The expression cassette of claim 22, wherein the non-viral vector is selected from the group consisting of naked DNA, naked RNA, plasmids, inorganic particles, lipid particles, polymer-based vectors, or chitosan-based formulations.

25. The expression cassette of claim 24, wherein the viral vector is a recombinant parvovirus, a recombinant lentivirus, a recombinant retrovirus, a recombinant adenovirus.

26. A plasmid comprising the expression cassette of any one of claims 12 to 24, optionally wherein the expression cassette is flanked by AAV 5 'itrs and AAV 3' itrs.

27. A host cell comprising the expression cassette of any one of claims 12 to 24 or the plasmid of claim 25.

28. A pharmaceutical composition comprising the rAAV according to any one of claims 1 to 11 or the expression cassette according to any one of claims 12 to 24, and a pharmaceutically acceptable carrier.

29. A method of treating a human subject diagnosed with GLA deficiency (fabry disease), the method comprising administering to the subject the rAAV of any one of claims 1-11, the expression cassette of any one of claims 12-24, or the pharmaceutical composition of claim 27.

30. The rAAV according to any one of claims 1 to 11, the expression cassette according to any one of claims 12 to 24 or the pharmaceutical composition according to claim 27 for use in the treatment of GLA deficiency (fabry disease).

31. The rAAV according to any one of claims 1 to 11, the expression cassette according to any one of claims 12 to 24 or the pharmaceutical composition according to claim 27 for use in the manufacture of a medicament for the treatment of GLA deficiency (fabry disease).