WO2024044697A2 - Compositions and methods for treatment of fabry disease - Google Patents
Compositions and methods for treatment of fabry disease Download PDFInfo
- Publication number
- WO2024044697A2 WO2024044697A2 PCT/US2023/072836 US2023072836W WO2024044697A2 WO 2024044697 A2 WO2024044697 A2 WO 2024044697A2 US 2023072836 W US2023072836 W US 2023072836W WO 2024044697 A2 WO2024044697 A2 WO 2024044697A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cells
- acid sequence
- nucleic acid
- seq
- engineered
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 118
- 238000011282 treatment Methods 0.000 title abstract description 20
- 208000024720 Fabry Disease Diseases 0.000 title abstract description 17
- 239000000203 mixture Substances 0.000 title abstract description 14
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 289
- 210000003719 b-lymphocyte Anatomy 0.000 claims abstract description 267
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 242
- 230000001225 therapeutic effect Effects 0.000 claims abstract description 153
- 210000004027 cell Anatomy 0.000 claims description 210
- 150000007523 nucleic acids Chemical group 0.000 claims description 189
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 138
- 101710163270 Nuclease Proteins 0.000 claims description 94
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 90
- 108020005004 Guide RNA Proteins 0.000 claims description 86
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 83
- 210000003720 plasmablast Anatomy 0.000 claims description 59
- 210000004180 plasmocyte Anatomy 0.000 claims description 51
- 101000718525 Homo sapiens Alpha-galactosidase A Proteins 0.000 claims description 45
- 108020004414 DNA Proteins 0.000 claims description 39
- 230000008685 targeting Effects 0.000 claims description 35
- 101001000998 Homo sapiens Protein phosphatase 1 regulatory subunit 12C Proteins 0.000 claims description 26
- 102100035620 Protein phosphatase 1 regulatory subunit 12C Human genes 0.000 claims description 24
- 238000012217 deletion Methods 0.000 claims description 22
- 230000037430 deletion Effects 0.000 claims description 22
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 claims description 17
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 claims description 17
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 14
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 14
- 108060003951 Immunoglobulin Proteins 0.000 claims description 12
- 102000043404 human GLA Human genes 0.000 claims description 12
- 102000018358 immunoglobulin Human genes 0.000 claims description 12
- 238000004520 electroporation Methods 0.000 claims description 11
- 239000002105 nanoparticle Substances 0.000 claims description 6
- 239000013603 viral vector Substances 0.000 claims description 6
- 102000053602 DNA Human genes 0.000 claims description 4
- 102000005840 alpha-Galactosidase Human genes 0.000 abstract description 135
- 108010030291 alpha-Galactosidase Proteins 0.000 abstract description 135
- 230000000694 effects Effects 0.000 abstract description 70
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 101000718529 Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) Alpha-galactosidase Proteins 0.000 abstract 1
- NNISLDGFPWIBDF-MPRBLYSKSA-N alpha-D-Gal-(1->3)-beta-D-Gal-(1->4)-D-GlcNAc Chemical compound O[C@@H]1[C@@H](NC(=O)C)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@@H](CO)O1 NNISLDGFPWIBDF-MPRBLYSKSA-N 0.000 abstract 1
- 235000018102 proteins Nutrition 0.000 description 148
- 108091033409 CRISPR Proteins 0.000 description 79
- 102000039446 nucleic acids Human genes 0.000 description 55
- 108020004707 nucleic acids Proteins 0.000 description 55
- 239000002773 nucleotide Substances 0.000 description 39
- 241000699670 Mus sp. Species 0.000 description 37
- 125000003729 nucleotide group Chemical group 0.000 description 36
- 108090000765 processed proteins & peptides Proteins 0.000 description 33
- 102100026277 Alpha-galactosidase A Human genes 0.000 description 31
- 235000001014 amino acid Nutrition 0.000 description 29
- 102000004196 processed proteins & peptides Human genes 0.000 description 26
- 238000010354 CRISPR gene editing Methods 0.000 description 25
- 230000014509 gene expression Effects 0.000 description 25
- 229920001184 polypeptide Polymers 0.000 description 25
- 125000006850 spacer group Chemical group 0.000 description 25
- 210000001744 T-lymphocyte Anatomy 0.000 description 24
- 238000010362 genome editing Methods 0.000 description 24
- 229940024606 amino acid Drugs 0.000 description 23
- 150000001413 amino acids Chemical class 0.000 description 22
- 238000010453 CRISPR/Cas method Methods 0.000 description 21
- 102000004389 Ribonucleoproteins Human genes 0.000 description 21
- 108010081734 Ribonucleoproteins Proteins 0.000 description 21
- 102000040430 polynucleotide Human genes 0.000 description 21
- 108091033319 polynucleotide Proteins 0.000 description 21
- 239000002157 polynucleotide Substances 0.000 description 21
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 20
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 17
- 210000001519 tissue Anatomy 0.000 description 16
- 238000012546 transfer Methods 0.000 description 16
- 239000013598 vector Substances 0.000 description 16
- 238000001727 in vivo Methods 0.000 description 15
- 238000003780 insertion Methods 0.000 description 15
- 230000037431 insertion Effects 0.000 description 15
- 238000006467 substitution reaction Methods 0.000 description 15
- 230000004048 modification Effects 0.000 description 14
- 238000012986 modification Methods 0.000 description 14
- 230000010354 integration Effects 0.000 description 13
- 238000002560 therapeutic procedure Methods 0.000 description 13
- 102000004190 Enzymes Human genes 0.000 description 12
- 108090000790 Enzymes Proteins 0.000 description 12
- 230000027455 binding Effects 0.000 description 12
- 230000005782 double-strand break Effects 0.000 description 12
- 208000024908 graft versus host disease Diseases 0.000 description 12
- 238000003786 synthesis reaction Methods 0.000 description 12
- 108020004705 Codon Proteins 0.000 description 11
- 102000004127 Cytokines Human genes 0.000 description 10
- 108090000695 Cytokines Proteins 0.000 description 10
- 241000702421 Dependoparvovirus Species 0.000 description 10
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 10
- 238000003556 assay Methods 0.000 description 10
- 230000015572 biosynthetic process Effects 0.000 description 10
- 210000002950 fibroblast Anatomy 0.000 description 10
- 238000000338 in vitro Methods 0.000 description 10
- 210000005259 peripheral blood Anatomy 0.000 description 10
- 239000011886 peripheral blood Substances 0.000 description 10
- 239000000126 substance Substances 0.000 description 10
- 208000024891 symptom Diseases 0.000 description 10
- 238000010361 transduction Methods 0.000 description 10
- 230000026683 transduction Effects 0.000 description 10
- 229940035893 uracil Drugs 0.000 description 10
- 239000011701 zinc Substances 0.000 description 10
- 229910052725 zinc Inorganic materials 0.000 description 10
- 230000004568 DNA-binding Effects 0.000 description 9
- 102100030704 Interleukin-21 Human genes 0.000 description 9
- 239000000427 antigen Substances 0.000 description 9
- 108091007433 antigens Proteins 0.000 description 9
- 102000036639 antigens Human genes 0.000 description 9
- 208000035475 disorder Diseases 0.000 description 9
- 238000009826 distribution Methods 0.000 description 9
- 230000002255 enzymatic effect Effects 0.000 description 9
- 108010074108 interleukin-21 Proteins 0.000 description 9
- 230000006780 non-homologous end joining Effects 0.000 description 9
- 230000003285 pharmacodynamic effect Effects 0.000 description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 8
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 8
- 108010073062 Transcription Activator-Like Effectors Proteins 0.000 description 8
- 238000007792 addition Methods 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 8
- 201000010099 disease Diseases 0.000 description 8
- 230000006870 function Effects 0.000 description 8
- 210000004976 peripheral blood cell Anatomy 0.000 description 8
- 230000028327 secretion Effects 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 238000001890 transfection Methods 0.000 description 8
- 102100027207 CD27 antigen Human genes 0.000 description 7
- 108010042407 Endonucleases Proteins 0.000 description 7
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 description 7
- 108010002350 Interleukin-2 Proteins 0.000 description 7
- 102000000588 Interleukin-2 Human genes 0.000 description 7
- -1 alpha- Chemical class 0.000 description 7
- 230000037396 body weight Effects 0.000 description 7
- 230000034431 double-strand break repair via homologous recombination Effects 0.000 description 7
- 238000001802 infusion Methods 0.000 description 7
- 230000002085 persistent effect Effects 0.000 description 7
- 238000013518 transcription Methods 0.000 description 7
- 230000035897 transcription Effects 0.000 description 7
- 208000009329 Graft vs Host Disease Diseases 0.000 description 6
- 241000193996 Streptococcus pyogenes Species 0.000 description 6
- 238000013459 approach Methods 0.000 description 6
- 210000004899 c-terminal region Anatomy 0.000 description 6
- 230000000295 complement effect Effects 0.000 description 6
- 239000002299 complementary DNA Substances 0.000 description 6
- 238000000684 flow cytometry Methods 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 6
- 238000002955 isolation Methods 0.000 description 6
- 230000002688 persistence Effects 0.000 description 6
- 230000008439 repair process Effects 0.000 description 6
- 230000003248 secreting effect Effects 0.000 description 6
- 102100031585 ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Human genes 0.000 description 5
- 241001655883 Adeno-associated virus - 1 Species 0.000 description 5
- 241000972680 Adeno-associated virus - 6 Species 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 5
- 108010029697 CD40 Ligand Proteins 0.000 description 5
- 102100032937 CD40 ligand Human genes 0.000 description 5
- 101000777636 Homo sapiens ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Proteins 0.000 description 5
- 102000003812 Interleukin-15 Human genes 0.000 description 5
- 108090000172 Interleukin-15 Proteins 0.000 description 5
- 108090001005 Interleukin-6 Proteins 0.000 description 5
- 102000004889 Interleukin-6 Human genes 0.000 description 5
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 5
- 108091034117 Oligonucleotide Proteins 0.000 description 5
- 238000004422 calculation algorithm Methods 0.000 description 5
- 230000003750 conditioning effect Effects 0.000 description 5
- 238000007847 digital PCR Methods 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 210000001806 memory b lymphocyte Anatomy 0.000 description 5
- 230000035772 mutation Effects 0.000 description 5
- 238000005457 optimization Methods 0.000 description 5
- 210000000056 organ Anatomy 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 4
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 4
- 102000004533 Endonucleases Human genes 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000010459 TALEN Methods 0.000 description 4
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 230000009471 action Effects 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 4
- 230000001419 dependent effect Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000002641 enzyme replacement therapy Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 4
- 239000008194 pharmaceutical composition Substances 0.000 description 4
- 108020003175 receptors Proteins 0.000 description 4
- 102000005962 receptors Human genes 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 210000003462 vein Anatomy 0.000 description 4
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 3
- 102100031780 Endonuclease Human genes 0.000 description 3
- 101150014526 Gla gene Proteins 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 3
- 101100005713 Homo sapiens CD4 gene Proteins 0.000 description 3
- 102000003814 Interleukin-10 Human genes 0.000 description 3
- 108090000174 Interleukin-10 Proteins 0.000 description 3
- 241000713666 Lentivirus Species 0.000 description 3
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 108010017070 Zinc Finger Nucleases Proteins 0.000 description 3
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 230000024245 cell differentiation Effects 0.000 description 3
- 210000000349 chromosome Anatomy 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000012636 effector Substances 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 229960000890 hydrocortisone Drugs 0.000 description 3
- 230000001976 improved effect Effects 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 238000001990 intravenous administration Methods 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 238000010172 mouse model Methods 0.000 description 3
- 229940021182 non-steroidal anti-inflammatory drug Drugs 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 230000036470 plasma concentration Effects 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000010561 standard procedure Methods 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 230000002459 sustained effect Effects 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 2
- VHRSUDSXCMQTMA-PJHHCJLFSA-N 6alpha-methylprednisolone Chemical compound C([C@@]12C)=CC(=O)C=C1[C@@H](C)C[C@@H]1[C@@H]2[C@@H](O)C[C@]2(C)[C@@](O)(C(=O)CO)CC[C@H]21 VHRSUDSXCMQTMA-PJHHCJLFSA-N 0.000 description 2
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 description 2
- 201000004384 Alopecia Diseases 0.000 description 2
- 108091008875 B cell receptors Proteins 0.000 description 2
- 108091079001 CRISPR RNA Proteins 0.000 description 2
- 241000282994 Cervidae Species 0.000 description 2
- 102000019034 Chemokines Human genes 0.000 description 2
- 108010012236 Chemokines Proteins 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 102000007644 Colony-Stimulating Factors Human genes 0.000 description 2
- 108010071942 Colony-Stimulating Factors Proteins 0.000 description 2
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 2
- 230000033616 DNA repair Effects 0.000 description 2
- 102000003951 Erythropoietin Human genes 0.000 description 2
- 108090000394 Erythropoietin Proteins 0.000 description 2
- 108010008165 Etanercept Proteins 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 108091029865 Exogenous DNA Proteins 0.000 description 2
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 2
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 2
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 description 2
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 description 2
- 108010000521 Human Growth Hormone Proteins 0.000 description 2
- 102000002265 Human Growth Hormone Human genes 0.000 description 2
- 239000000854 Human Growth Hormone Substances 0.000 description 2
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 2
- HEFNNWSXXWATRW-UHFFFAOYSA-N Ibuprofen Chemical compound CC(C)CC1=CC=C(C(C)C(O)=O)C=C1 HEFNNWSXXWATRW-UHFFFAOYSA-N 0.000 description 2
- 108010061833 Integrases Proteins 0.000 description 2
- UETNIIAIRMUTSM-UHFFFAOYSA-N Jacareubin Natural products CC1(C)OC2=CC3Oc4c(O)c(O)ccc4C(=O)C3C(=C2C=C1)O UETNIIAIRMUTSM-UHFFFAOYSA-N 0.000 description 2
- 102100020880 Kit ligand Human genes 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- 108060001084 Luciferase Proteins 0.000 description 2
- 239000005089 Luciferase Substances 0.000 description 2
- 102000009151 Luteinizing Hormone Human genes 0.000 description 2
- 108010073521 Luteinizing Hormone Proteins 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 108010006519 Molecular Chaperones Proteins 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- CMWTZPSULFXXJA-UHFFFAOYSA-N Naproxen Natural products C1=C(C(C)C(O)=O)C=CC2=CC(OC)=CC=C21 CMWTZPSULFXXJA-UHFFFAOYSA-N 0.000 description 2
- 108010025020 Nerve Growth Factor Proteins 0.000 description 2
- 102000007072 Nerve Growth Factors Human genes 0.000 description 2
- 241000288906 Primates Species 0.000 description 2
- 230000026279 RNA modification Effects 0.000 description 2
- 208000035977 Rare disease Diseases 0.000 description 2
- 108010039445 Stem Cell Factor Proteins 0.000 description 2
- 108091027544 Subgenomic mRNA Proteins 0.000 description 2
- 102000036693 Thrombopoietin Human genes 0.000 description 2
- 108010041111 Thrombopoietin Proteins 0.000 description 2
- 102000011923 Thyrotropin Human genes 0.000 description 2
- 108010061174 Thyrotropin Proteins 0.000 description 2
- 108010009583 Transforming Growth Factors Proteins 0.000 description 2
- 102000009618 Transforming Growth Factors Human genes 0.000 description 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 2
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 2
- 102100040247 Tumor necrosis factor Human genes 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 239000002260 anti-inflammatory agent Substances 0.000 description 2
- 229940121363 anti-inflammatory agent Drugs 0.000 description 2
- 238000003149 assay kit Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 238000002659 cell therapy Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 229940047120 colony stimulating factors Drugs 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000004590 computer program Methods 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 229960004397 cyclophosphamide Drugs 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 229960003957 dexamethasone Drugs 0.000 description 2
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 2
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 2
- 210000005069 ears Anatomy 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 229940105423 erythropoietin Drugs 0.000 description 2
- 229940126864 fibroblast growth factor Drugs 0.000 description 2
- 229940028334 follicle stimulating hormone Drugs 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 239000003862 glucocorticoid Substances 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 208000024963 hair loss Diseases 0.000 description 2
- 230000003676 hair loss Effects 0.000 description 2
- 238000011577 humanized mouse model Methods 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 229960002591 hydroxyproline Drugs 0.000 description 2
- 229960001680 ibuprofen Drugs 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 239000000367 immunologic factor Substances 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000015788 innate immune response Effects 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- VHOGYURTWQBHIL-UHFFFAOYSA-N leflunomide Chemical compound O1N=CC(C(=O)NC=2C=CC(=CC=2)C(F)(F)F)=C1C VHOGYURTWQBHIL-UHFFFAOYSA-N 0.000 description 2
- 229960000681 leflunomide Drugs 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 229940040129 luteinizing hormone Drugs 0.000 description 2
- 210000003712 lysosome Anatomy 0.000 description 2
- 230000001868 lysosomic effect Effects 0.000 description 2
- 229960000485 methotrexate Drugs 0.000 description 2
- 229960004584 methylprednisolone Drugs 0.000 description 2
- LXBIFEVIBLOUGU-DPYQTVNSSA-N migalastat Chemical group OC[C@H]1NC[C@H](O)[C@@H](O)[C@H]1O LXBIFEVIBLOUGU-DPYQTVNSSA-N 0.000 description 2
- 229950007469 migalastat Drugs 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 229960002009 naproxen Drugs 0.000 description 2
- CMWTZPSULFXXJA-VIFPVBQESA-N naproxen Chemical compound C1=C([C@H](C)C(O)=O)C=CC2=CC(OC)=CC=C21 CMWTZPSULFXXJA-VIFPVBQESA-N 0.000 description 2
- 229940046166 oligodeoxynucleotide Drugs 0.000 description 2
- 239000000816 peptidomimetic Substances 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 2
- 229960005205 prednisolone Drugs 0.000 description 2
- OIGNJSKKLXVSLS-VWUMJDOOSA-N prednisolone Chemical compound O=C1C=C[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OIGNJSKKLXVSLS-VWUMJDOOSA-N 0.000 description 2
- 229960004618 prednisone Drugs 0.000 description 2
- XOFYZVNMUHMLCC-ZPOLXVRWSA-N prednisone Chemical compound O=C1C=C[C@]2(C)[C@H]3C(=O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 XOFYZVNMUHMLCC-ZPOLXVRWSA-N 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000000750 progressive effect Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000010473 stable expression Effects 0.000 description 2
- 229960001940 sulfasalazine Drugs 0.000 description 2
- NCEXYHBECQHGNR-QZQOTICOSA-N sulfasalazine Chemical compound C1=C(O)C(C(=O)O)=CC(\N=N\C=2C=CC(=CC=2)S(=O)(=O)NC=2N=CC=CC=2)=C1 NCEXYHBECQHGNR-QZQOTICOSA-N 0.000 description 2
- NCEXYHBECQHGNR-UHFFFAOYSA-N sulfasalazine Natural products C1=C(O)C(C(=O)O)=CC(N=NC=2C=CC(=CC=2)S(=O)(=O)NC=2N=CC=CC=2)=C1 NCEXYHBECQHGNR-UHFFFAOYSA-N 0.000 description 2
- 229940037128 systemic glucocorticoids Drugs 0.000 description 2
- 230000002123 temporal effect Effects 0.000 description 2
- 230000005030 transcription termination Effects 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 230000001052 transient effect Effects 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- LJRDOKAZOAKLDU-UDXJMMFXSA-N (2s,3s,4r,5r,6r)-5-amino-2-(aminomethyl)-6-[(2r,3s,4r,5s)-5-[(1r,2r,3s,5r,6s)-3,5-diamino-2-[(2s,3r,4r,5s,6r)-3-amino-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-6-hydroxycyclohexyl]oxy-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl]oxyoxane-3,4-diol;sulfuric ac Chemical compound OS(O)(=O)=O.N[C@@H]1[C@@H](O)[C@H](O)[C@H](CN)O[C@@H]1O[C@H]1[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](N)C[C@@H](N)[C@@H]2O)O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)N)O[C@@H]1CO LJRDOKAZOAKLDU-UDXJMMFXSA-N 0.000 description 1
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- TVYLLZQTGLZFBW-ZBFHGGJFSA-N (R,R)-tramadol Chemical compound COC1=CC=CC([C@]2(O)[C@H](CCCC2)CN(C)C)=C1 TVYLLZQTGLZFBW-ZBFHGGJFSA-N 0.000 description 1
- FUFLCEKSBBHCMO-UHFFFAOYSA-N 11-dehydrocorticosterone Natural products O=C1CCC2(C)C3C(=O)CC(C)(C(CC4)C(=O)CO)C4C3CCC2=C1 FUFLCEKSBBHCMO-UHFFFAOYSA-N 0.000 description 1
- BRMWTNUJHUMWMS-UHFFFAOYSA-N 3-Methylhistidine Natural products CN1C=NC(CC(N)C(O)=O)=C1 BRMWTNUJHUMWMS-UHFFFAOYSA-N 0.000 description 1
- AGFIRQJZCNVMCW-UAKXSSHOSA-N 5-bromouridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(Br)=C1 AGFIRQJZCNVMCW-UAKXSSHOSA-N 0.000 description 1
- 229940117976 5-hydroxylysine Drugs 0.000 description 1
- 108010059616 Activins Proteins 0.000 description 1
- 102000005606 Activins Human genes 0.000 description 1
- 241000282979 Alces alces Species 0.000 description 1
- 235000002198 Annona diversifolia Nutrition 0.000 description 1
- 241000272517 Anseriformes Species 0.000 description 1
- 108010005853 Anti-Mullerian Hormone Proteins 0.000 description 1
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 1
- 241000203069 Archaea Species 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 230000003844 B-cell-activation Effects 0.000 description 1
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- VOVIALXJUBGFJZ-KWVAZRHASA-N Budesonide Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@@H]2[C@@H]1[C@@H]1C[C@H]3OC(CCC)O[C@@]3(C(=O)CO)[C@@]1(C)C[C@@H]2O VOVIALXJUBGFJZ-KWVAZRHASA-N 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 102100021809 Chorionic somatomammotropin hormone 1 Human genes 0.000 description 1
- MFYSYFVPBJMHGN-ZPOLXVRWSA-N Cortisone Chemical compound O=C1CC[C@]2(C)[C@H]3C(=O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 MFYSYFVPBJMHGN-ZPOLXVRWSA-N 0.000 description 1
- MFYSYFVPBJMHGN-UHFFFAOYSA-N Cortisone Natural products O=C1CCC2(C)C3C(=O)CC(C)(C(CC4)(O)C(=O)CO)C4C3CCC2=C1 MFYSYFVPBJMHGN-UHFFFAOYSA-N 0.000 description 1
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 1
- 108010036949 Cyclosporine Proteins 0.000 description 1
- 102220605874 Cytosolic arginine sensor for mTORC1 subunit 2_D10A_mutation Human genes 0.000 description 1
- 150000008574 D-amino acids Chemical class 0.000 description 1
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- XUIIKFGFIJCVMT-GFCCVEGCSA-N D-thyroxine Chemical compound IC1=CC(C[C@@H](N)C(O)=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-GFCCVEGCSA-N 0.000 description 1
- 230000008265 DNA repair mechanism Effects 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 108010087819 Fc receptors Proteins 0.000 description 1
- 102000009109 Fc receptors Human genes 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- 241000699694 Gerbillinae Species 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000006771 Gonadotropins Human genes 0.000 description 1
- 108010086677 Gonadotropins Proteins 0.000 description 1
- 241000282575 Gorilla Species 0.000 description 1
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 241000282596 Hylobatidae Species 0.000 description 1
- 102000012960 Immunoglobulin kappa-Chains Human genes 0.000 description 1
- 108010090227 Immunoglobulin kappa-Chains Proteins 0.000 description 1
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical class O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
- 108090001117 Insulin-Like Growth Factor II Proteins 0.000 description 1
- 102100037852 Insulin-like growth factor I Human genes 0.000 description 1
- 102100025947 Insulin-like growth factor II Human genes 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 102000012330 Integrases Human genes 0.000 description 1
- 102000006992 Interferon-alpha Human genes 0.000 description 1
- 108010047761 Interferon-alpha Proteins 0.000 description 1
- 102000003996 Interferon-beta Human genes 0.000 description 1
- 108090000467 Interferon-beta Proteins 0.000 description 1
- 102000008070 Interferon-gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 102000000589 Interleukin-1 Human genes 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 108090000177 Interleukin-11 Proteins 0.000 description 1
- 102000003815 Interleukin-11 Human genes 0.000 description 1
- 102000013462 Interleukin-12 Human genes 0.000 description 1
- 108010065805 Interleukin-12 Proteins 0.000 description 1
- 102000004125 Interleukin-1alpha Human genes 0.000 description 1
- 108010082786 Interleukin-1alpha Proteins 0.000 description 1
- 108010002386 Interleukin-3 Proteins 0.000 description 1
- 102000000646 Interleukin-3 Human genes 0.000 description 1
- 102000004388 Interleukin-4 Human genes 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 108010002616 Interleukin-5 Proteins 0.000 description 1
- 102100039897 Interleukin-5 Human genes 0.000 description 1
- 108010002586 Interleukin-7 Proteins 0.000 description 1
- 102100021592 Interleukin-7 Human genes 0.000 description 1
- 108090001007 Interleukin-8 Proteins 0.000 description 1
- 102000004890 Interleukin-8 Human genes 0.000 description 1
- 108010002335 Interleukin-9 Proteins 0.000 description 1
- 102000000585 Interleukin-9 Human genes 0.000 description 1
- 102000015696 Interleukins Human genes 0.000 description 1
- 108010063738 Interleukins Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- LRQKBLKVPFOOQJ-YFKPBYRVSA-N L-norleucine Chemical compound CCCC[C@H]([NH3+])C([O-])=O LRQKBLKVPFOOQJ-YFKPBYRVSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 125000000510 L-tryptophano group Chemical group [H]C1=C([H])C([H])=C2N([H])C([H])=C(C([H])([H])[C@@]([H])(C(O[H])=O)N([H])[*])C2=C1[H] 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 241000282842 Lama glama Species 0.000 description 1
- 102000008072 Lymphokines Human genes 0.000 description 1
- 108010074338 Lymphokines Proteins 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 208000015439 Lysosomal storage disease Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102000013967 Monokines Human genes 0.000 description 1
- 108010050619 Monokines Proteins 0.000 description 1
- DTERQYGMUDWYAZ-ZETCQYMHSA-N N(6)-acetyl-L-lysine Chemical compound CC(=O)NCCCC[C@H]([NH3+])C([O-])=O DTERQYGMUDWYAZ-ZETCQYMHSA-N 0.000 description 1
- JDHILDINMRGULE-LURJTMIESA-N N(pros)-methyl-L-histidine Chemical compound CN1C=NC=C1C[C@H](N)C(O)=O JDHILDINMRGULE-LURJTMIESA-N 0.000 description 1
- JJIHLJJYMXLCOY-BYPYZUCNSA-N N-acetyl-L-serine Chemical compound CC(=O)N[C@@H](CO)C(O)=O JJIHLJJYMXLCOY-BYPYZUCNSA-N 0.000 description 1
- PYUSHNKNPOHWEZ-YFKPBYRVSA-N N-formyl-L-methionine Chemical compound CSCC[C@@H](C(O)=O)NC=O PYUSHNKNPOHWEZ-YFKPBYRVSA-N 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- BRUQQQPBMZOVGD-XFKAJCMBSA-N Oxycodone Chemical compound O=C([C@@H]1O2)CC[C@@]3(O)[C@H]4CC5=CC=C(OC)C2=C5[C@@]13CCN4C BRUQQQPBMZOVGD-XFKAJCMBSA-N 0.000 description 1
- 241000282579 Pan Species 0.000 description 1
- 102000003982 Parathyroid hormone Human genes 0.000 description 1
- 108090000445 Parathyroid hormone Proteins 0.000 description 1
- 102000015731 Peptide Hormones Human genes 0.000 description 1
- 108010038988 Peptide Hormones Proteins 0.000 description 1
- 241000286209 Phasianidae Species 0.000 description 1
- 108010003044 Placental Lactogen Proteins 0.000 description 1
- 239000000381 Placental Lactogen Substances 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 241000282405 Pongo abelii Species 0.000 description 1
- 108010076181 Proinsulin Proteins 0.000 description 1
- 102000003946 Prolactin Human genes 0.000 description 1
- 108010057464 Prolactin Proteins 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108090000103 Relaxin Proteins 0.000 description 1
- 102000003743 Relaxin Human genes 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 108091028113 Trans-activating crRNA Proteins 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- 102400001320 Transforming growth factor alpha Human genes 0.000 description 1
- 101800004564 Transforming growth factor alpha Proteins 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 1
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 1
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 1
- 241000589634 Xanthomonas Species 0.000 description 1
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 229960001138 acetylsalicylic acid Drugs 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000000488 activin Substances 0.000 description 1
- 229960002964 adalimumab Drugs 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 229940035676 analgesics Drugs 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 239000000730 antalgic agent Substances 0.000 description 1
- 229940124599 anti-inflammatory drug Drugs 0.000 description 1
- 239000000868 anti-mullerian hormone Substances 0.000 description 1
- 210000000628 antibody-producing cell Anatomy 0.000 description 1
- 102000025171 antigen binding proteins Human genes 0.000 description 1
- 108091000831 antigen binding proteins Proteins 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229940009098 aspartate Drugs 0.000 description 1
- AUJRCFUBUPVWSZ-XTZHGVARSA-M auranofin Chemical compound CCP(CC)(CC)=[Au]S[C@@H]1O[C@H](COC(C)=O)[C@@H](OC(C)=O)[C@H](OC(C)=O)[C@H]1OC(C)=O AUJRCFUBUPVWSZ-XTZHGVARSA-M 0.000 description 1
- 229960005207 auranofin Drugs 0.000 description 1
- LMEKQMALGUDUQG-UHFFFAOYSA-N azathioprine Chemical compound CN1C=NC([N+]([O-])=O)=C1SC1=NC=NC2=C1NC=N2 LMEKQMALGUDUQG-UHFFFAOYSA-N 0.000 description 1
- 229960002170 azathioprine Drugs 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 229960002537 betamethasone Drugs 0.000 description 1
- UREBDLICKHMUKA-DVTGEIKXSA-N betamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-DVTGEIKXSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 108010006025 bovine growth hormone Proteins 0.000 description 1
- 229960004436 budesonide Drugs 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- UHBYWPGGCSDKFX-UHFFFAOYSA-N carboxyglutamic acid Chemical compound OC(=O)C(N)CC(C(O)=O)C(O)=O UHBYWPGGCSDKFX-UHFFFAOYSA-N 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 108700010039 chimeric receptor Proteins 0.000 description 1
- 229960001265 ciclosporin Drugs 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- ALEXXDVDDISNDU-JZYPGELDSA-N cortisol 21-acetate Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@@](C(=O)COC(=O)C)(O)[C@@]1(C)C[C@@H]2O ALEXXDVDDISNDU-JZYPGELDSA-N 0.000 description 1
- 229960004544 cortisone Drugs 0.000 description 1
- 230000000139 costimulatory effect Effects 0.000 description 1
- 229940111134 coxibs Drugs 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 239000003255 cyclooxygenase 2 inhibitor Substances 0.000 description 1
- 229930182912 cyclosporin Natural products 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 230000008260 defense mechanism Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 229940124447 delivery agent Drugs 0.000 description 1
- YSMODUONRAFBET-UHFFFAOYSA-N delta-DL-hydroxylysine Natural products NCC(O)CCC(N)C(O)=O YSMODUONRAFBET-UHFFFAOYSA-N 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- NAGJZTKCGNOGPW-UHFFFAOYSA-K dioxido-sulfanylidene-sulfido-$l^{5}-phosphane Chemical compound [O-]P([O-])([S-])=S NAGJZTKCGNOGPW-UHFFFAOYSA-K 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 229940073621 enbrel Drugs 0.000 description 1
- YSMODUONRAFBET-UHNVWZDZSA-N erythro-5-hydroxy-L-lysine Chemical compound NC[C@H](O)CC[C@H](N)C(O)=O YSMODUONRAFBET-UHNVWZDZSA-N 0.000 description 1
- 229960000403 etanercept Drugs 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000012997 ficoll-paque Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 229940076085 gold Drugs 0.000 description 1
- 239000002622 gonadotropin Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940048921 humira Drugs 0.000 description 1
- 229960001067 hydrocortisone acetate Drugs 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- XXSMGPRMXLTPCZ-UHFFFAOYSA-N hydroxychloroquine Chemical compound ClC1=CC=C2C(NC(C)CCCN(CCO)CC)=CC=NC2=C1 XXSMGPRMXLTPCZ-UHFFFAOYSA-N 0.000 description 1
- 229960004171 hydroxychloroquine Drugs 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 125000001841 imino group Chemical group [H]N=* 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 229960000598 infliximab Drugs 0.000 description 1
- 239000000893 inhibin Substances 0.000 description 1
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 102000006495 integrins Human genes 0.000 description 1
- 108010044426 integrins Proteins 0.000 description 1
- 229940047124 interferons Drugs 0.000 description 1
- 229940047122 interleukins Drugs 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 241001515942 marmosets Species 0.000 description 1
- 238000013178 mathematical model Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000002483 medication Methods 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- HPNSFSBZBAHARI-UHFFFAOYSA-N micophenolic acid Natural products OC1=C(CC=C(C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-UHFFFAOYSA-N 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- DYKFCLLONBREIL-KVUCHLLUSA-N minocycline Chemical compound C([C@H]1C2)C3=C(N(C)C)C=CC(O)=C3C(=O)C1=C(O)[C@@]1(O)[C@@H]2[C@H](N(C)C)C(O)=C(C(N)=O)C1=O DYKFCLLONBREIL-KVUCHLLUSA-N 0.000 description 1
- 229960004023 minocycline Drugs 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229940014456 mycophenolate Drugs 0.000 description 1
- HPNSFSBZBAHARI-RUDMXATFSA-N mycophenolic acid Chemical compound OC1=C(C\C=C(/C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-RUDMXATFSA-N 0.000 description 1
- 229960003940 naproxen sodium Drugs 0.000 description 1
- CDBRNDSHEYLDJV-FVGYRXGTSA-M naproxen sodium Chemical compound [Na+].C1=C([C@H](C)C([O-])=O)C=CC2=CC(OC)=CC=C21 CDBRNDSHEYLDJV-FVGYRXGTSA-M 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 239000000041 non-steroidal anti-inflammatory agent Substances 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 238000002515 oligonucleotide synthesis Methods 0.000 description 1
- 208000038009 orphan disease Diseases 0.000 description 1
- 230000002138 osteoinductive effect Effects 0.000 description 1
- 229960002085 oxycodone Drugs 0.000 description 1
- 229960005489 paracetamol Drugs 0.000 description 1
- 239000000199 parathyroid hormone Substances 0.000 description 1
- 229960001319 parathyroid hormone Drugs 0.000 description 1
- 229960001639 penicillamine Drugs 0.000 description 1
- 239000000813 peptide hormone Substances 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- PTMHPRAIXMAOOB-UHFFFAOYSA-N phosphoramidic acid Chemical compound NP(O)(O)=O PTMHPRAIXMAOOB-UHFFFAOYSA-N 0.000 description 1
- BZQFBWGGLXLEPQ-REOHCLBHSA-N phosphoserine Chemical compound OC(=O)[C@@H](N)COP(O)(O)=O BZQFBWGGLXLEPQ-REOHCLBHSA-N 0.000 description 1
- 244000000003 plant pathogen Species 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 229920001451 polypropylene glycol Polymers 0.000 description 1
- 230000023603 positive regulation of transcription initiation, DNA-dependent Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 229940097325 prolactin Drugs 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 108010087851 prorelaxin Proteins 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 229940116176 remicade Drugs 0.000 description 1
- 230000008263 repair mechanism Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000003118 sandwich ELISA Methods 0.000 description 1
- JRPHGDYSKGJTKZ-UHFFFAOYSA-K selenophosphate Chemical compound [O-]P([O-])([O-])=[Se] JRPHGDYSKGJTKZ-UHFFFAOYSA-K 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 230000004797 therapeutic response Effects 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 1
- 229940034208 thyroxine Drugs 0.000 description 1
- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 description 1
- 230000005100 tissue tropism Effects 0.000 description 1
- 229960004380 tramadol Drugs 0.000 description 1
- TVYLLZQTGLZFBW-GOEBONIOSA-N tramadol Natural products COC1=CC=CC([C@@]2(O)[C@@H](CCCC2)CN(C)C)=C1 TVYLLZQTGLZFBW-GOEBONIOSA-N 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 229960005294 triamcinolone Drugs 0.000 description 1
- GFNANZIMVAIWHM-OBYCQNJPSA-N triamcinolone Chemical compound O=C1C=C[C@]2(C)[C@@]3(F)[C@@H](O)C[C@](C)([C@@]([C@H](O)C4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 GFNANZIMVAIWHM-OBYCQNJPSA-N 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 230000024883 vasodilation Effects 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 229920003169 water-soluble polymer Polymers 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
- C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01022—Alpha-galactosidase (3.2.1.22)
Definitions
- Fabry disease is an incurable, progressive lysosomal storage disease characterized by mutations in the GLA gene, which results in the absence or decreased function of the encoded alpha-galactosidase A (a-GAL) enzyme, leading to the accumulation of Globotriaosylceramide (Gb3) throughout the body.
- a-GAL alpha-galactosidase A
- Gb3 Globotriaosylceramide
- Oral chaperone therapy is available to a subset of patients who have amenable GLA-mutations; however, the majority of patients are treated with enzyme replacement therapy (ERT): a bi-weekly infusion of recombinant a-GAL.
- ERT enzyme replacement therapy
- a-GAL ERT therapy is a lifelong commitment typically requiring infusions that can last for several hours, frequently cause acute infusion reactions, results in highly variable plasma a-GAL concentrations due to short a-GAL half-life, and places a large burden on financial and other resources within the health care system. Due to these challenges, long-term compliance is difficult. A need for novel and more universal treatment of Fabry disease is remains.
- the invention relates to a therapeutic protein comprising a modified human a-GAL protein, wherein the modified human a-GAL protein has been modified by, a deletion of two carboxy terminal leucine residues of a wild-type human a- GAL protein; and a replacement of a signal peptide of said wild-type human a-GAL protein with an immunoglobulin(Ig) signal peptide.
- the signal peptide is a heavy chain Ig signal peptide. In various embodiments, the signal peptide is a light chain Ig signal peptide. In various embodiments, the therapeutic protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments, the therapeutic protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments, the therapeutic protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments, the therapeutic protein comprises an amino acid sequence that is selected from: SEQ ID Nos: 2-4.
- the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the modified human a- GAL protein is encoded by a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the modified human a-GAL protein is encoded by a nucleic acid sequence is selected from: SEQ ID Nos: 6-8, 16-17 or 20-21.
- the invention relates to an engineered human B cell comprising a therapeutic protein, wherein the therapeutic protein comprises a modified human GLA wherein the modified human a-GAL protein has been modified by, a deletion of two carboxy terminal leucine residues of a wild-type human a-GAL protein; and a replacement of a signal peptide of said wild-type human a-GAL protein with an immunoglobulin(Ig) signal peptide.
- the therapeutic protein comprises a modified human GLA wherein the modified human a-GAL protein has been modified by, a deletion of two carboxy terminal leucine residues of a wild-type human a-GAL protein; and a replacement of a signal peptide of said wild-type human a-GAL protein with an immunoglobulin(Ig) signal peptide.
- the engineered B cell further comprises a nucleic acid sequence encoding said therapeutic protein, and wherein said nucleic acid sequence has been inserted into either the AAVS1 or the human ROSA26 locus.
- the signal peptide is a heavy chain Ig signal peptide.
- the signal peptide is a light chain Ig signal peptide.
- the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 85% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4.
- the modified human a- GAL protein is encoded by a nucleic acid sequence that is at least 90% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments, the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 95% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments, the modified human a-GAL protein is encoded by a nucleic acid sequence that is selected from: SEQ ID Nos: 2-4.
- the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the modified human a- GAL protein is encoded by a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the modified human a-GAL protein is encoded by a nucleic acid sequence that is selected from: SEQ ID Nos: 6-8, 16-17 or 20-21.
- the gene has been inserted into the human AAVS1 locus. In various embodiments, the gene has been inserted into the human ROSA26 locus. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells, wherein at least 20% of the population of cells express said therapeutic protein. In various embodiments said B cell is CD20+. In various embodiments said B cell is CD20-. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells, wherein a majority of said cells are CD20+. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells, wherein more than 80 percent of said cells are CD20-.
- said B cell is a plasmablast.
- the invention relates to a population of cells comprising a plurality of engineered B cells, wherein a majority of said cells are plasmablasts.
- said cell is a plasma cell.
- a population of cells comprising a plurality of engineered B cells, wherein a majority of said cells are plasma cells.
- the invention relates to a method of producing an engineered B cell expressing a therapeutic protein, the method comprising delivering to a human B cell: an RNA-guided nuclease; a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein, wherein the therapeutic protein comprises a human a-GAL protein.
- the RNA-guided nuclease and gRNA are delivered to the B cell as an RNP. In various embodiments, the RNA-guided nuclease and gRNA are delivered to the B cell as a nanoparticle. In various embodiments, the RNA-guided nuclease and gRNA are delivered to the B cell via electroporation. In various embodiments, the construct delivered to the B cell using a viral vector. In various embodiments, the construct delivered to the B cell as double stranded DNA. In various embodiments, the RNA-guided nuclease comprises the amino acid sequence of SEQ ID No. 14 or SEQ ID No. 24.
- the RNA-guided nuclease comprises the amino acid sequence of SEQ ID NO. 15.
- the targeting construct comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 9-13 18-19 and 25-29.
- said engineered B cell is CD20-. In various embodiments, said engineered B cell is CD20+. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells, wherein a majority of said cells are CD20-. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells, wherein more than 80 percent of said cells are CD20+. In various embodiments, said engineered B cell is a plasmablast. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells, wherein a majority of said cells are plasmablasts. In various embodiments, said engineered B cell is a plasma cells. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells, wherein a majority of said cells are plasma cells.
- the invention relates to a method of producing an engineered B cell expressing a therapeutic protein, the method comprising delivering to a human B cell: a RNA-guided nuclease; a gRNA targeting the AAVS1 gene, wherein the gRNA comprises the nucleic acid sequence of SEQ ID NO. 14, 15 or 24; and a construct comprising a nucleic acid sequence encoding a therapeutic protein and a left and right homology arm; wherein the construct comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 9-13 and 18-19, wherein said engineered B cell expresses said therapeutic protein.
- the invention relates to a method of treating a patient in need thereof comprising administering to said patient an engineered human B cell, wherein the engineered human B cell has been edited to express a therapeutic protein encoding a human a-GAL protein.
- the therapeutic protein comprises a modified human a-GAL protein, wherein the two carboxy terminal leucine residues of the human a-GAL protein have been deleted; and the GLA signal peptide has been replaced by an immunoglobulin (Ig) signal peptide.
- the signal peptide is a heavy chain Ig signal peptide.
- the signal peptide is a light chain Ig signal peptide.
- the therapeutic protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments, the therapeutic protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments, the therapeutic protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments, the therapeutic protein comprises an amino acid sequence that is selected from: SEQ ID Nos: 2-4. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21.
- the therapeutic protein is encoded by a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20- 21.
- the gene encoding the therapeutic protein has been inserted into the human AAVS1 locus. In various embodiments, the gene encoding the therapeutic protein has been inserted into the human ROSA26 locus. In various embodiments, at least 20% of the population of cells express said therapeutic protein. In various embodiments, said population of cells comprise cells that are CD20+. In various embodiments, said population of cells comprise cells that are CD20+. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells according to any one of claims 54-64, wherein a majority of said cells are CD20+.
- the invention relates to a population of cells comprising a plurality of engineered B cells according to any one of claims 54-64, wherein more than 80 percent of said cells are CD20-.
- said engineered B cell is a plasmablast.
- the invention relates to a population of cells comprising a plurality of engineered B cells, wherein a majority of said cells are plasmablasts.
- said engineered B cell is a plasma cells.
- the invention relates to a population of cells comprising a plurality of engineered B cells, wherein a majority of said cells are plasma cells.
- the invention relates to, a method of treating a patient in need thereof comprising administering to said patient an engineered human B cell, wherein said human B cell comprises a therapeutic protein, encoded by a gene, wherein the therapeutic protein comprises a modified human a-GAL protein, wherein the two carboxy terminal leucine residues of the human GLA protein have been deleted; and the GLA signal peptide has been replaced by an immunoglobulin (Ig) signal peptide.
- Ig immunoglobulin
- the nucleic acid sequence encoding said therapeutic protein has been inserted into either the AAVS1 or the human ROSA26 locus.
- the signal peptide is a heavy chain Ig signal peptide.
- the signal peptide is a light chain Ig signal peptide.
- the therapeutic protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4.
- the therapeutic protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4.
- the therapeutic protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments, the therapeutic protein comprises an amino acid sequence that is selected from: SEQ ID Nos: 2-4. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21.
- the therapeutic protein is encoded by a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21.
- the gene has been inserted into the human AAVS1 locus. In various embodiments, the gene has been inserted into the human ROSA26 locus. In various embodiments, at least 20% of the population of engineered B cells, express said therapeutic protein. In various embodiments, said population of cells comprise cells that are CD20+. In various embodiments, said population of cells comprise cells that are CD20-. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells wherein a majority of said cells are CD20+. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells, wherein more than 80 percent of said cells are CD20-.
- said engineered B cell is a plasmablast. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells, wherein a majority of said cells are plasmablasts. In various embodiments, said engineered B cell is a plasma cells. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells, wherein a majority of said cells are plasma cells.
- the invention relates to a method of treating a patient in need thereof comprising administering to said patient a therapeutic protein comprising a modified human a-GAL protein, wherein the two carboxy terminal leucine residues have been deleted; and the GLA signal peptide has been replaced by an immunoglobulin (Ig) signal peptide.
- a therapeutic protein comprising a modified human a-GAL protein, wherein the two carboxy terminal leucine residues have been deleted; and the GLA signal peptide has been replaced by an immunoglobulin (Ig) signal peptide.
- Ig immunoglobulin
- the signal peptide is a heavy chain Ig signal peptide. In various embodiments, the signal peptide is a light chain Ig signal peptide. In various embodiments, the therapeutic protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments, the therapeutic protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments, the therapeutic protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments the therapeutic protein comprises an amino acid sequence that is selected from: SEQ ID Nos: 2-4.
- the therapeutic protein is encoded by a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20- 21.
- FIG. 1 shows a schematic of two insertion sites for targeted integration as per certain embodiments of the present invention.
- FIG. 1 A shows insertion of the GLA gene at the human AAVS1 locus on chromosome 19. The insert size of this targeting construct is approximately 3700 bp.
- FIG. IB shows insertion of the GLA gene at the human ROSA26 locus on chromosome 3.
- FIG 2A shows a-GAL activity after insertion at the AAVS1 locus.
- FIG. 2B shows a-GAL activity after insertion at the ROSA26 locus.
- peripheral blood B cells become activated and some differentiate towards plasmablasts (PB) and plasma cells (PC).
- PB plasmablasts
- PC plasma cells
- peripheral blood B cells edited to express human GLA showed significantly enhanced a-GAL activity, indicating integration.
- FIG. 3 shows a-GAL activity in activated B-cells and peripheral blood cells differentiated toward PBs and PCs that have been edited to express various versions of the a- GAL protein
- FIG. 5 shows a-GAL activity in activated B cells and peripheral blood cells differentiated toward PBs and PCs.
- Human B cells were activated for 3 days in a media containing CD40L, CpG, IL2, IL 10, IL 15 and IL21.
- Gene editing (1140 GLA with LL truncation and heavy chain signal peptide (SEQ ID No. 11)) was performed at day 3 after isolation and cells were expanded in the same media for an additional 6 days. Subsequently, expanded B cells were culture in a media containing IL2, IL6, IL10, IL15, IL21 for 3 days, to obtain a mixture of plasmablasts and B cells.
- FIG. 6 shows a-GAL activity in edited human peripheral blood cells differentiated toward PBs.
- B cells edited to express human GLA (labeled in FIG. 6 as “Edited” followed by the construct number) showed significantly enhanced a-GAL activity.
- FIG. 7A shows the mechanism by which a-GAL enters cells. Phosphorylated a- GAL binds to M6P receptors (M6P-R) and is internalized in the lysosome.
- FIG. 7B shows restoration of intracellular a-GAL levels in fibroblasts derived from Fabry patients. Fibroblasts cultured with concentrated supernatant from 293 cells secreting either WT (1002, SEQ ID No. 5) a-GAL or truncated a-GAL (1067, SEQ ID No. 7) showed increased a-GAL activity when compared with both WT and Fabry fibroblasts alone.
- FIG. 8 shows expression of a-GAL protein from edited B cells in vivo.
- FIG. 8 A shows a-GAL enzyme activity measured from plasma samples collected 3 days after B cells transfer and every 10 days up to 100 days.
- FIG. 8B shows a-GAL protein expression using an ELISA from plasma taken from the same NSG mice on days 3, 10 and 20.
- FIG. 9 shows gene editing efficiency (FIG. 9A) and on-target GLA integration (FIG. 9B in peripheral blood human B cells gene edited using a Ribonucleoproteins (RNP) targeting an AAV1 locus delivered into B cells via nucleofection and GLA cDNA via recombinant AAV6 (rAAV6) transduction.
- RNP Ribonucleoproteins
- rAAV6 recombinant AAV6
- FIG. 10 shows that optimized single guide RNA mediate efficient on-target gene editing with no off-target events.
- FIG. 11 shows the effect of codon optimization on a-GAL activity in vitro.
- Human B cells were edited using various GLA-cDNA sequences (FIG. 11 A (SEQ ID No. 11) and FIG. 1 IB (SEQ ID No. 25)) and cultured for 12 days. At 9 and 12 days, cell culture supernatants were collected, and cell counts and viability were assessed. Functional, secreted a-GAL was quantified using an activity assay and results were normalized to cell counts.
- FIG. 12 shows a guide-SEQ analysis of genomic DNA isolated from Human B cells.
- On-target/off-target sites were identified by incorporation of doublestranded oligodeoxynucleotides (dsODNs).
- dsODNs doublestranded oligodeoxynucleotides
- FIG. 13 A shows an overview of various ex vivo B cell engineering and culture procedures.
- FIG. 14 shows B cell lineages after in vitro conditioning.
- FIG. 14A shows various cell populations, memory B cells (CD27+IgD-), plasmablasts (PBs) (CD27+CD38+ CD20-), and plasma cells (PCs) (CD27+CD38+ CD20- CD 138+) populations monitored using Flow cytometry.
- FIG. 14B shows PrimeFlow analysis of GLA transcripts in B cell populations.
- FIG. 15 shows sustained, supraphysiologic a-GAL plasma levels in vivo.
- Human B cells were engineered and cultured as described.
- GLA-edited B cells were adoptively transferred into NSG mice pre-conditioned with adoptive transfer of CD4+ T cells prior to adoptive transfer of GLA-edited B cells.
- FIG. 16 shows adoptively transferred genetically engineered B cells engrafted in mice in vivo.
- Engineered human B cells were adoptively transferred into CD4+ T cell humanized NSG mice.
- Representative luciferase image shows early engraftment of the engineered cells primarily in the bone marrow (FIG. 16A).
- FIG. 17 shows characterization of the effect of editing on patient-derived engineered B cells from Fabry patient’s PBMCs.
- B cells were isolated from Fabry patient’s PBMCs via CD 19+ positive selection, and activated for 3 days in media containing CD40L, CpG, IL-2, IL- 10, IL- 15 and IL-21.
- Gene editing was performed on day 3 of culture using Cas9/sgRNA delivered via electroporation and by transduction via AAV6 of active enzyme GLA (1606) followed by an additional 7 days of culture.
- Engineered B cells were expanded in the same media described above for an additional 7 days.
- FIG. 17A shows that the Fabry edited B cells were capable of expansion and growth.
- FIG. 17A shows that the Fabry edited B cells were capable of expansion and growth.
- FIG. 17B shows that the Fabry B cell had successful integration.
- FIG. 17C shows that GLA-Edited Fabry cells showed increased secretion of a-GAL active enzyme when compared to non-edited controls.
- FIG. 18 shows systemic levels of a-GAL increase over time in both total CD4+ and memory CD4+ T cells mouse models. NSG mice were humanized via adoptive transfer of either total human CD4+ cells or memory human CD4+ cells together with edited B cells (1140, SEQ ID No. 11) and a-GAL activity was assessed. Peripheral blood B cells edited to express human GLA showed significantly enhanced a-GAL activity in both mouse models (Memory CD4+ and Total CD4+) when compared to mice injected with saline.
- FIG. 19 shows memory CD4+ T cells mouse model does not show any symptoms of GvHD.
- NSG mice were humanized as described in FIG 18 were assessed for bodyweight and GVHD symptoms e.g., hair loss, redness ears/extremities, hunched posture).
- mice infused with Memory CD4+ T cells together with edited B cells (1140, SEQ ID No. 11) showed no difference in body weight or GVHD symptoms.
- mice infused with Total CD4+ T cells together with edited B cells (1140, SEQ ID No. 11) showed reduction in body weight and/or exhibited GVHD symptoms (FIG. 19C).
- FIG. 20 shows a-GAL activity in edited human PBs using various codon optimized constructs in activated B cells and peripheral blood cells differentiated toward PBs in Donor 1 (FIG. 20A) and Donor 2 (FIG. 20B).
- B cells edited to express human GLA (“Edited”) showed significantly enhanced a-GAL activity.
- Construct 1482 is the original cDNA sequence (1140, SEQ ID No. 11). Of the five codon optimized constructs (1588, 1598, 1599, 1600 and 1606), construct 1606 and 1599 showed enhanced a-GAL activity in PB cells when compared to activated B cells and compared to construct 1482.
- Various embodiments disclosed herein provide for autologous human B-cells that have been gene- edited to express a functional a-GAL protein and will provide long-term stable expression of the enzyme in vivo.
- the advantages of such a cellular therapy include: (i) decreased frequency of infusion (e.g., every 12 months instead of every two weeks); (ii) decreased rate and severity of acute infusion reactions; (iii) improved steady state expression of enzyme which may reduce the risk of immunogenicity and enable better clinical outcome; (iv) potential improved patient compliance and therefore better clinical outcome.
- polynucleotide includes both singlestranded and double-stranded nucleotide polymers.
- the nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide.
- Said modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2’, 3 ’-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphoro-diselenoate, phosphoro-anilothioate, phoshoraniladate and phosphoroamidate.
- base modifications such as bromouridine and inosine derivatives
- ribose modifications such as 2’, 3 ’-dideoxyribose
- internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphoro-diselenoate, phosphoro-anilothioate, phoshoraniladate and phosphoroamidate.
- oligonucleotide refers to a polynucleotide comprising 200 or fewer nucleotides. Oligonucleotides can be single stranded or double stranded, e.g., for use in the construction of a mutant gene. Oligonucleotides can be sense or antisense oligonucleotides. An oligonucleotide can include a label, including a radiolabel, a fluorescent label, a hapten or an antigenic label, for detection assays. Oligonucleotides can be used, for example, as PCR primers, cloning primers or hybridization probes.
- control sequence refers to a polynucleotide sequence that can affect the expression and processing of coding sequences to which it is ligated.
- control sequences for prokaryotes can include a promoter, a ribosomal binding site, and a transcription termination sequence.
- control sequences for eukaryotes can include promoters comprising one or a plurality of recognition sites for transcription factors, transcription enhancer sequences, and transcription termination sequence.
- Control sequences can include leader sequences (signal peptides) and/or fusion partner sequences.
- operably linked means that the components to which the term is applied are in a relationship that allows them to carry out their inherent functions under suitable conditions.
- vector means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage or virus) used to transfer protein coding information into a host cell.
- expression vector or “expression construct” refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control (in conjunction with the host cell) expression of one or more heterologous coding regions operatively linked thereto.
- An expression construct can include, but is not limited to, sequences that affect or control transcription, translation, and, if introns are present, affect RNA splicing of a coding region operably linked thereto.
- the term “host cell” refers to a cell that has been transformed, or is capable of being transformed, with a nucleic acid sequence and thereby expresses a gene of interest.
- the term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent cell, so long as the gene of interest is present.
- transformation refers to a change in a cell’s genetic characteristics, and a cell has been transformed when it has been modified to contain new DNA or RNA.
- a cell is transformed where it is genetically modified from its native state by introducing new genetic material via transfection, transduction, or other genetic engineering techniques.
- the transforming DNA can recombine with that of the cell by physically integrating into a chromosome of the cell, or can be maintained transiently as an episomal element without being replicated, or can replicate independently as a plasmid.
- a cell is considered to have been “stably transformed” when the transforming DNA is replicated with the division of the cell.
- transfection refers to the uptake of foreign or exogenous DNA by a cell.
- transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology, 1973, 52:456; Sambrook et al., Molecular Cloning: A Laboratory Manual, 2001, supra; Davis et al., Basic Methods in Molecular Biology, 1986, Elsevier; Chu c/ a/., 1981, Gene, 13: 197.
- transduction refers to the process whereby foreign DNA is introduced into a cell via viral vector. See, e.g., Jones et al., Genetics: Principles and Analysis, 1998, Boston: Jones & Bartlett Publ.
- polypeptide or “protein” refer to a macromolecule having the amino acid sequence of a protein, including deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence.
- polypeptide and protein specifically encompass antigen-binding molecules, antibodies, or sequences that have deletions from, additions to, and/or substitutions of one or more amino acid of antigenbinding protein.
- polypeptide fragment refers to a polypeptide that has an aminoterminal deletion, a carboxyl-terminal deletion, and/or an internal deletion as compared with the full-length native protein. Such fragments can also contain modified amino acids as compared with the native protein.
- Useful polypeptide fragments include immunologically functional fragments of antigen-binding molecules.
- a “variant” of a polypeptide comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to another polypeptide sequence.
- Variants include fusion proteins.
- identity refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent identity” means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (i.e., an “algorithm”).
- the sequences being compared are typically aligned in a way that gives the largest match between the sequences.
- One example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., Nucl. Acid Res., 1984, 12, 387; Genetics Computer Group, University of Wisconsin, Madison, Wis.).
- GAP is used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined.
- the sequences are aligned for optimal matching of their respective amino acid or nucleotide (the “matched span”, as determined by the algorithm).
- a standard comparison matrix (see, e.g., Dayhoff et al., 1978, Atlas of Protein Sequence and Structure, 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A., 89, 10915-10919 for the BLO-SUM 62 comparison matrix) is also used by the algorithm.
- the twenty conventional (e.g., naturally occurring) amino acids and their abbreviations follow conventional usage. See, e.g., Immunology A Synthesis (2nd Edition, Golub and Green, Eds., Sinauer Assoc., Sunderland, Mass. (1991)), which is incorporated herein by reference for any purpose.
- Stereoisomers e.g., D-amino acids
- unnatural amino acids such as alpha-, alpha-di substituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids can also be suitable components for polypeptides of the present invention.
- Examples of unconventional amino acids include: 4-hydroxyproline, gamma-carboxy-glutamate, epsilon- N,N,N-trimethyllysine, e-N-acetyllysine, O-phosphoserine, N-acetylserine, N- formylmethionine, 3-methylhistidine, 5-hydroxylysine, sigma. -N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline).
- the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.
- Naturally occurring residues can be divided into classes based on common side chain properties: a) hydrophobic: norleucine, Met, Ala, Vai, Leu, He; b) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; c) acidic: Asp, Glu; d) basic: His, Lys, Arg; e) residues that influence chain orientation: Gly, Pro; and f) aromatic: Trp, Tyr, Phe.
- non-conservative substitutions can involve the exchange of a member of one of these classes for a member from another class.
- the hydropathic index of amino acids can be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics.
- derivative refers to a molecule that includes a chemical modification other than an insertion, deletion, or substitution of amino acids (or nucleic acids).
- derivatives comprise covalent modifications, including, but not limited to, chemical bonding with polymers, lipids, or other organic or inorganic moieties.
- a chemically modified antigen-binding molecule can have a greater circulating half-life than an antigen-binding molecule that is not chemically modified.
- a derivative antigen-binding molecule is covalently modified to include one or more water-soluble polymer attachments, including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.
- Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed “peptide mimetics” or “peptidomimetics.” Fauchere, J. L., 1986, Adv. Drug Res., 1986, 15, 29; Veber, D. F. & Freidinger, R. M., 1985, Trends in Neuroscience, 8, 392-396; and Evans, B. E., et al., 1987, J. Med. Chem., 30, 1229-1239, which are incorporated herein by reference for any purpose.
- therapeutically effective amount refers to a quantity or amount of an agent (e.g., therapeutic cell compositions, immune cells or other therapeutic agent) sufficient to achieve a desired therapeutic effect or response in a subject.
- agent e.g., therapeutic cell compositions, immune cells or other therapeutic agent
- patient and “subject” are used interchangeably and include human and non-human animal subjects as well as those with formally diagnosed disorders, those without formally recognized disorders, those receiving medical attention, those at risk of developing the disorders, etc.
- treatment includes therapeutic treatments, prophylactic treatments, and applications in which one reduces the risk that a subject will develop a disorder or other risk factor. Treatment does not require the complete curing of a disorder and encompasses embodiments in which one reduces symptoms or underlying risk factors.
- prevent does not require the 100% elimination of the possibility of an event. Rather, it denotes that the likelihood of the occurrence of the event has been reduced in the presence of the compound or method.
- Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection).
- Enzymatic reactions and purification techniques can be performed according to manufacturer’s specifications or as commonly accomplished in the art or as described herein.
- the foregoing techniques and procedures can be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.
- the term “substantially” or “essentially” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% higher compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
- the terms “essentially the same” or “substantially the same” refer to a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is about the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
- the terms “substantially free of’ and “essentially free of’ are used interchangeably, and when used to describe a composition, such as a cell population or culture media, refer to a composition that is free of a specified substance, such as, 95% free, 96% free, 97% free, 98% free, 99% free of the specified substance, or is undetectable as measured by conventional means. Similar meaning can be applied to the term “absence of,” where referring to the absence of a particular substance or component of a composition. [0069] As used herein, the term “appreciable” refers to a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length or an event that is readily detectable by one or more standard methods.
- not-appreciable and “not appreciable” and equivalents refer to a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length or an event that is not readily detectable or undetectable by standard methods. In one embodiment, an event is not appreciable if it occurs less than 5%, 4%, 3%, 2%, 1%, 0.1%, 0.001%, or less of the time.
- the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
- the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 15%, 10%, 5% or 1%, or any intervening ranges thereof.
- introducing refers to a process that comprises contacting a cell with a polynucleotide, polypeptide, or small molecule.
- An introducing step may also comprise microinjection of polynucleotides or polypeptides into the cell, use of liposomes to deliver polynucleotides or polypeptides into the cell, or fusion of polynucleotides or polypeptides to cell permeable moi eties to introduce them into a cell.
- the engineered B cell comprises a therapeutic protein to be delivered to a patient in need thereof.
- therapeutic protein means any protein that may contribute to the treatment, reduction of symptoms, prevention or cure of a disease or disorder in a patient.
- the therapeutic protein may be suitable for treatment of a rare disease or an orphan disease, where said therapy can be achieved by the replacement of a particular protein and/or enzyme.
- a therapeutic protein may include but is not limited to an enzyme, a ligand, a naturally occurring, engineered and/or chimeric receptor, a cytokine or a chemokine.
- said therapeutic protein is a protein for the treatment of Fabry disease.
- the therapeutic protein is a-galactosidase (a-GAL).
- the therapeutic protein is human a-GAL.
- the therapeutic protein comprises the amino acid sequence of SEQ ID NO. 1.
- the therapeutic protein is at least 75%, 80%, 85%, 90% or 95% identical to the amino acid sequence of SEQ ID NO. 1.
- the therapeutic protein is encoded by a nucleic acid sequence comprising SEQ ID NO. 5.
- the therapeutic protein is encoded by a nucleic acid sequence comprising a sequence that is at least 75%, 80%, 85%, 90% or 95% identical to the nucleic acid sequence of SEQ ID NO. 5.
- the therapeutic protein comprises the human a-GAL protein, wherein the two leucine residues at the carboxy terminus have been deleted.
- the therapeutic protein comprises the amino acid sequence of SEQ ID NO. 2.
- the therapeutic protein is at least 75%, 80%, 85%, 90% or 95% identical to the amino acid sequence of SEQ ID NO. 2.
- the therapeutic protein is encoded by a nucleic acid sequence comprising SEQ ID NO. 6.
- the therapeutic protein is encoded by a nucleic acid sequence comprising a sequence that is at least 75%, 80%, 85%, 90% or 95% identical to the nucleic acid sequence of SEQ ID NO. 6.
- the therapeutic protein is a human a-GAL protein, wherein the two leucine residues at the carboxy terminus have been deleted, and the natural human GLA signal peptide has been replaced with an immunoglobulin (Ig) signal peptide.
- Ig immunoglobulin
- Modification to include an Ig signal peptide directs the a-GAL enzyme towards the secretory pathway, thereby promoting secretion of the expressed therapeutic protein.
- the heavy chain Ig signal peptide is used.
- the therapeutic protein comprises the amino acid sequence of SEQ ID NO. 3. In various embodiments, the therapeutic protein is at least 75%, 80%, 85%, 90% or 95% identical to the amino acid sequence of SEQ ID NO. 3. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence comprising SEQ ID NO. 7. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence comprising a sequence that is at least 75%, 80%, 85%, 90% or 95% identical to the nucleic acid sequence of SEQ ID NO. 7.
- the kappa light chain Ig signal peptide is used.
- the therapeutic protein comprises the amino acid sequence of SEQ ID NO. 4. In various embodiments, the therapeutic protein is at least 75%, 80%, 85%, 90% or 95% identical to the amino acid sequence of SEQ ID NO. 4. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence comprising SEQ ID NO. 8. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence comprising a sequence that is at least 75%, 80%, 85%, 90% or 95% identical to the nucleic acid sequence of SEQ ID NO. 8.
- the therapeutic protein is expressed by engineering a population of B cells to express said therapeutic protein.
- B cell refers to an immune cell (e.g., a white blood cell or leukocyte) that expresses a B cell receptor (BCR) or produces antibodies.
- B cell includes any B cell lineage cell type that is derived from a B cell including memory B cells, plasma cells (PCs) and plasmablasts (PBs).
- a plasmablast is an intermediate, transitional stage cell type that arises during differentiation of activated B cells. It is the immediate precursor to a plasma cell.
- Plasma cells are the terminal, fully differentiated form resulting from B cell differentiation.
- the primary function of plasma cells is the robust synthesis and secretion of antibodies.
- Plasma cells have a highly developed secretory system to support a high level of protein synthesis and secretion. Although some plasma cells are short-lived, others can persist much longer.
- Antigen-induced B cell activation and differentiation can also result in memory B cells, which can persist for years. Upon a secondary encounter with the same antigen, memory B cells can also proliferate and differentiate into plasma cells.
- the embodiments disclosed herein provide long-term stable expression of the enzyme in vivo.
- the embodiments disclosed herein leverage the B cell’s intrinsic protein secretion machinery and its long lifetime in vivo to express therapeutic proteins for treatment of multiple diseases including Fabry disease.
- the disclosure relates to a population of cells comprising engineered human B cells, wherein human B cells express a therapeutic protein, whose gene has been inserted into either the human AAVS1 or the human ROSA26 locus, wherein the therapeutic protein comprises the human GLA protein.
- said population of cells are for treatment of a patient suffering from Fabry disease.
- the population of cells express a therapeutic protein that is a-galactosidase (a-GAL).
- the population of cells express a therapeutic protein that is wild type human a-GAL.
- the population of cells express a therapeutic protein that comprises the amino acid sequence of SEQ ID NO. 1.
- the population of cells express a therapeutic protein that is at least 75%, 80%, 85%, 90% or 95% identical to the amino acid sequence of SEQ ID NO. 1.
- the population of cells express a therapeutic protein that is encoded by a nucleic acid sequence comprising SEQ ID NO. 5.
- the population of cells express a therapeutic protein that is encoded by a nucleic acid sequence comprising a sequence that is at least 75%, 80%, 85%, 90% or 95% identical to the nucleic acid sequence of SEQ ID NO. 5.
- the population of cells express a therapeutic protein comprising the human a-GAL protein, wherein the two leucine residues at the carboxy terminus have been deleted.
- the population of cells express a therapeutic protein that comprises the amino acid sequence of SEQ ID NO. 2.
- the population of cells express a therapeutic protein that is at least 75%, 80%, 85%, 90% or 95% identical to the amino acid sequence of SEQ ID NO. 2.
- the population of cells express a therapeutic protein that is encoded by a nucleic acid sequence comprising SEQ ID NO. 6.
- the population of cells express a therapeutic protein that is encoded by a nucleic acid sequence comprising a sequence that is at least 75%, 80%, 85%, 90% or 95% identical to the nucleic acid sequence of SEQ ID NO. 6.
- the population of cells express a therapeutic protein comprising the human a-GAL protein, wherein the two leucine residues at the carboxy terminus have been deleted, and the natural human a-GAL signal peptide has been replaced with an immunoglobulin (Ig) signal peptide.
- Ig immunoglobulin
- Modification to include an Ig signal peptide directs the a-GAL enzyme towards the secretory pathway, thereby promoting secretion of the expressed therapeutic protein.
- the heavy chain Ig signal peptide is used.
- the population of cells express a therapeutic protein that comprises the amino acid sequence of SEQ ID NO. 3.
- the population of cells express a therapeutic protein that is at least 75%, 80%, 85%, 90% or 95% identical to the amino acid sequence of SEQ ID NO. 3.
- the population of cells express a therapeutic protein that is encoded by a nucleic acid sequence comprising SEQ ID NO. 7.
- the population of cells express a therapeutic protein that is encoded by a nucleic acid sequence comprising a sequence that is at least 75%, 80%, 85%, 90% or 95% identical to the nucleic acid sequence of SEQ ID NO. 7.
- the kappa light chain Ig signal peptide is used.
- the population of cells express a therapeutic protein that comprises the amino acid sequence of SEQ ID NO. 4.
- the population of cells express a therapeutic protein that is at least 75%, 80%, 85%, 90% or 95% identical to the amino acid sequence of SEQ ID NO. 4.
- the population of cells express a therapeutic protein that is encoded by a nucleic acid sequence comprising SEQ ID NO. 8.
- the population of cells express a therapeutic protein that is encoded by a nucleic acid sequence comprising a sequence that is at least 75%, 80%, 85%, 90% or 95% identical to the nucleic acid sequence of SEQ ID NO. 8.
- a certain percentage of the population of cells will express said therapeutic protein. In various embodiments at least 20% of the population of cells express said therapeutic protein. In various embodiments at least 10%, 15%, 20%, 25%, 30%, 35% or 40% of the population of cells express said therapeutic protein.
- Gene editing is a type of genetic engineering in which nucleotide(s)/nucleic acid(s) is/are inserted, deleted, and/or substituted in a DNA sequence, such as in the genome of a targeted cell.
- Targeted gene editing enables insertion, deletion and/or substitution at pre-selected sites in the genome of a targeted cell (e.g., in a targeted gene or targeted DNA sequence).
- the endogenous gene comprising the affected sequence may be knocked-out or knocked-down due to the sequence alteration.
- Targeted editing may be used to disrupt endogenous gene expression.
- “Targeted integration” refers to a process involving insertion of one or more exogenous sequences, with or without deletion of an endogenous sequence at the insertion site. Targeted integration can result from targeted gene editing when a donor template containing an exogenous sequence is present.
- a “disrupted gene” refers to a gene comprising an insertion, deletion, or substitution relative to an endogenous gene such that expression of a functional protein from the endogenous gene is reduced or inhibited.
- disrupting a gene refers to a method of inserting, deleting or substituting at least one nucleotide/nucleic acid in an endogenous gene such that expression of a functional protein from the endogenous gene is reduced or inhibited. Methods of disrupting a gene are known to those of skill in the art and described herein.
- Targeted editing can be achieved either through a nuclease-independent approach, or through a nuclease - dependent approach.
- nuclease-independent targeted editing approach homologous recombination is guided by homologous sequences flanking an exogenous polynucleotide to be introduced into an endogenous sequence through the enzymatic machinery of the host cell.
- the exogenous polynucleotide may introduce deletions, insertions or replacement of nucleotides in the endogenous sequence.
- nuclease - dependent approach can achieve targeted editing with higher frequency through the specific introduction of double strand breaks (DSBs) by specific rare - cutting nucleases (e.g., endonucleases).
- DSBs double strand breaks
- endonucleases e.g., endonucleases
- Such nuclease - dependent targeted editing also utilizes DNA repair mechanisms, for example, non - homologous end joining (NHEJ), which occurs in response to DSBs.
- NHEJ non - homologous end joining
- DNA repair by NHEJ often leads to random insertions or deletions (indels) of a small number of endogenous nucleotides.
- HDR homology directed repair
- Available endonucleases capable of introducing specific and targeted DSBs include, but are not limited to, zinc-finger nucleases (ZFN), meganucleases, transcription activator-like effector nucleases (TALEN), and RNA-guided CRISPR Cas9 nuclease (CRISPR/Cas9; Clustered Regular Interspaced Short Palindromic Repeats Associated 9). Additionally, DICE (dual integrase cassette exchange) system utilizing phiC31 and Bxbl integrases may also be used for targeted integration.
- ZFN zinc-finger nucleases
- TALEN transcription activator-like effector nucleases
- CRISPR/Cas9 Clustered Regular Interspaced Short Palindromic Repeats Associated 9
- DICE dual integrase cassette exchange
- ZFNs are targeted nucleases comprising a nuclease fused to a zinc finger DNA binding domain (ZFBD), which is a polypeptide domain that binds DNA in a sequence specific manner.
- ZFBD zinc finger DNA binding domain
- a zinc finger is a domain of about 30 amino acids within the zinc fingerbinding domain whose structure is stabilized through coordination of a zinc ion. Examples of zinc fingers include, but not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers.
- a designed zinc finger domain is a domain not occurring in nature whose design/composition results principally from rational criteria, e.g., application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. See, for example, U.S. Pat.
- a selected zinc finger domain is a domain not found in nature whose production results primarily from an empirical process such as phage display, interaction trap or hybrid selection.
- ZFNs are described in greater detail in U.S. Pat. Nos. 7,888,121 and 7,972,854. The most recognized example of a ZFN is a fusion of the Fokl nuclease with a zinc finger DNA binding domain.
- a TALEN is a targeted nuclease comprising a nuclease fused to a TAL effector DNA binding domain.
- a “transcription activator-like effector DNA binding domain”, “TAL effector DNA binding domain”, or “TALE DNA binding domain” is a polypeptide domain of TAL effector proteins that is responsible for binding of the TAL effector protein to DNA.
- TAL effector proteins are secreted by plant pathogens of the genus Xanthomonas during infection. These proteins enter the nucleus of the plant cell, bind effector-specific DNA sequences via their DNA binding domain, and activate gene transcription at these sequences via their transactivation domains.
- TAL effector DNA binding domain specificity depends on an effector - variable number of imperfect 34 amino acid repeats, which comprise polymorphisms at select repeat positions called repeat variable diresidues (RVD).
- RVD repeat variable diresidues
- TALENs are described in greater detail in US Patent Application No. 2011/0145940. The most recognized example of a TALEN in the art is a fusion polypeptide of the Fokl nuclease to a TAL effector DNA binding domain.
- targeted nucleases suitable for use as provided herein include, but are not limited to, Bxbl, phiC31, R4, PhiBTl, and WB/SPBc/TP901-l, whether used individually or in combination.
- targeted nucleases include naturally - occurring and recombinant nucleases, e.g., CRISPR/Cas9, restriction endonucleases, meganucleases homing endonucleases, and the like.
- the CRISPR-Cas9 system is a naturally-occurring defense mechanism in prokaryotes that has been repurposed as an RNA-guided DNA - targeting platform used for gene editing. It relies on the DNA nuclease Cas9, and two noncoding RNAs, crisprRNA (CrRNA) and trans-activating RNA (tracrRNA), to target the cleavage of DNA.
- PrRNA crisprRNA
- tracrRNA trans-activating RNA
- CRISPR is an abbreviation for Clustered Regularly Interspaced Short Palindromic Repeats, a family of DNA sequences found in the genomes of bacteria and archaea that contain fragments of DNA (spacer DNA) with similarity to foreign DNA previously exposed to the cell, for example, by viruses that have infected or attacked the prokaryote. These fragments of DNA are used by the prokaryote to detect and destroy similar foreign DNA upon reintroduction, for example, from similar viruses during subsequent attacks. Transcription of the CRISPR locus results in the formation of an RNA molecule comprising the spacer sequence, which associates with and targets Cas (CRISPR-associated) proteins able to recognize and cut the foreign, exogenous DNA. Numerous types and classes of CRISPR/Cas systems have been described (see, e.g., Koonin et al., (2017) Curr Opin Microbiol 37:67-78).
- crRNA drives sequence recognition and specificity of the CRISPR - Cas9 complex through Watson - Crick base pairing typically with a 20 nucleotide (nt) sequence in the target DNA. Changing the sequence of the 5 ' 20nt in the crRNA allows targeting of the CRISPR - Cas9 complex to specific loci.
- the CRISPR - Cas9 complex only binds DNA sequences that contain a sequence match to the first 20 nt of the crRNA, single - guide RNA (sgRNA), if the target sequence is followed by a specific short DNA motif (with the sequence NGG) referred to as a protospacer adjacent motif (PAM).
- sgRNA single - guide RNA
- PAM protospacer adjacent motif
- TracrRNA hybridizes with the 3' end of crRNA to form an RNA-duplex structure that is bound by the Cas9 endonuclease to form the catalytically active CRISPR-Cas9 complex, which can then cleave the target DNA.
- NHEJ non - homologous end - joining
- HDR homology - directed repair
- HDR uses a long stretch of homologous donor DNA, provided endogenously or exogenously, to repair the DSB with high fidelity.
- HDR is active only in dividing cells, and occurs at a relatively low frequency in most cell types.
- NHEJ is utilized as the repair operant.
- the Cas9 (CRISPR associated protein 9) endonuclease is from Streptococcus pyogenes, although other Cas9 homologs may be used. It should be understood, that wild - type Cas9 may be used or modified versions of Cas9 may be used (e.g., evolved versions of Cas9, or Cas9 orthologues or variants), as provided herein. In some embodiments, Cas9 may be substituted with another RNA- guided endonuclease, such as Cpfl (of a class II CRISPR/Cas system).
- Cpfl RNA- guided endonuclease
- the CRISPR/Cas system comprise components derived from a Type-1, Type-II, or Type-III system.
- Updated classification schemes for CRISPR/Cas loci define Class 1 and Class 2 CRISPR/Cas systems, having Types Ito V or VI (Makarova et al., (2015) Nat Rev Microbiol, 13(11):722-36; Shmakov etal., (2015)) Mol Cell, 60:385- 397).
- Class 2 CRISPR / Cas systems have single protein effectors.
- Cas proteins of Types II, V, and VI are single - protein, RNA - guided endonucleases, herein called “Class 2 Cas nucleases.”
- Class 2 Cas nucleases include, for example, Cas9, Cpfl, C2cl, C2c2, and C2c3 proteins.
- the Cpfl nuclease (Zetsche et al., (2015) Cell 163: 1-13) is homologous to Cas9, and contains a RuvC - like nuclease domain.
- the Cas nuclease is from a Type - II CRISPR/Cas system (e.g., a Cas9 protein from a CRISPR / Cas9 system).
- the Cas nuclease is from a Class 2 CRISPR Cas system (a single protein Cas nuclease such as a Cas9 protein or a Cpfl protein).
- the Cas9 and Cpfl family of proteins are enzymes with DNA endonuclease activity, and they can be directed to cleave a desired nucleic acid target by designing an appropriate guide RNA, as described further herein.
- a Cas nuclease may comprise more than one nuclease domain.
- a Cas9 nuclease may comprise at least one RuvC-like nuclease domain (e.g., Cpfl) and at least one HNH-like nuclease domain (e.g., Cas9).
- the Cas9 nuclease introduces a DSB in the target sequence.
- the Cas9 nuclease is modified to contain only one functional nuclease domain.
- the Cas9 nuclease is modified such that one of the nuclease domains is mutated or fully or partially deleted to reduce its nucleic acid cleavage activity.
- the Cas9 nuclease is modified to contain no functional RuvC-like nuclease domain.
- the Cas9 nuclease is modified to contain no functional HNH- like nuclease domain.
- the Cas9 nuclease is a nickase that is capable of introducing a single-stranded break (a “nick”) into the target sequence.
- a conserved amino acid within a Cas9 nuclease domain is substituted to reduce or alter a nuclease activity.
- the Cas nuclease nickase comprises an amino acid substitution in the RuvC- like nuclease domain.
- Exemplary amino acid substitutions in the RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 nuclease).
- the nickase comprises an amino acid substitution in the HNH-like nuclease domain.
- Exemplary amino acid substitutions in the HNH-like nuclease domain include E762A, H840A, N863 A, H983 A, and D986A (based on the S. pyogenes Cas9 nuclease).
- the Cas nuclease is from a Type-I CRISPR/Cas system.
- the Cas nuclease is a component of the Cascade complex of a Type-I CRISPR/Cas system.
- the Cas nuclease is a Cas3 nuclease.
- the Cas nuclease is derived from a Type-III CRISPR/Cas system.
- the Cas nuclease is derived from Type-IV CRISPR/Cas system.
- the Cas nuclease is derived from a Type-V CRISPR/Cas system.
- the Cas nuclease is derived from a Type- VI CRISPR/Cas system.
- the present disclosure provides a genome-targeting nucleic acid that can direct the activities of an associated polypeptide (e.g., a site-directed polypeptide) to a specific target sequence within a target nucleic acid.
- the genome-targeting nucleic acid can be an RNA.
- a genome-targeting RNA is referred to as a “guide RNA” or “gRNA” herein.
- a guide RNA comprises at least a spacer sequence that hybridizes to a target nucleic acid sequence of interest, and a CRISPR repeat sequence.
- the gRNA also comprises a second RNA called the tracrRNA sequence.
- the CRISPR repeat sequence and tracrRNA sequence hybridize to each other to form a duplex.
- the crRNA forms a duplex.
- the duplex binds a site-directed polypeptide, such that the guide RNA and site-direct polypeptide form a complex.
- the genome-targeting nucleic acid provides target specificity to the complex by virtue of its association with the site-directed polypeptide. The genome-targeting nucleic acid thus directs the activity of the site-directed polypeptide.
- each guide RNA is designed to include a spacer sequence complementary to its genomic target sequence. See Jinek et a/., Science, 337, 816-821 (2012) and Deltcheva et al., Nature, 471, 602-607 (2011).
- the genome-targeting nucleic acid e.g., gRNA
- the genome-targeting nucleic acid is a double-molecule guide RNA.
- the genome-targeting nucleic acid is a single-molecule guide RNA.
- a double-molecule guide RNA comprises two strands of RNA.
- the first strand comprises in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence and a minimum CRISPR repeat sequence.
- the second strand comprises a minimum tracrRNA sequence (complementary to the minimum CRISPR repeat sequence), a 3' tracrRNA sequence and an optional tracrRNA extension sequence.
- a single-molecule guide RNA in a Type II system comprises, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single-molecule guide linker, a minimum tracrRNA sequence, a 3' tracrRNA sequence and an optional tracrRNA extension sequence.
- the optional tracrRNA extension may comprise elements that contribute additional functionality (e.g., stability) to the guide RNA.
- the single-molecule guide linker links the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure.
- the optional tracrRNA extension comprises one or more hairpins.
- a single-molecule guide RNA in a Type V system comprises, in the 5' to 3' direction, a minimum CRISPR repeat sequence and a spacer sequence.
- the sgRNA comprises a 20 nucleotide spacer sequence at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA comprises a less than 20 nucleotide spacer sequence at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA comprises a more than 20 nucleotide spacer sequence at the 5' end of the sgRNA sequence.
- the sgRNA comprises comprise no uracil at the 3' end of the sgRNA sequence. In some embodiments, the sgRNA comprises comprise one or more uracil at the 3' end of the sgRNA sequence.
- the sgRNA can comprise 1 uracil (U) at the 3' end of the sgRNA sequence.
- the sgRNA can comprise 2 uracil (UU) at the 3' end of the sgRNA sequence.
- the sgRNA can comprise 3 uracil (UUU) at the 3' end of the sgRNA sequence.
- the sgRNA can comprise 4 uracil (UUUU) at the 3' end of the sgRNA sequence.
- the sgRNA can comprise 5 uracil (UUUUU) at the 3' end of the sgRNA sequence.
- the sgRNA can comprise 6 uracil (UUUUUU) at the 3' end of the sgRNA sequence.
- the sgRNA can comprise 7 uracil (UUUUUUU) at the 3' end of the sgRNA sequence.
- the sgRNA can comprise 8 uracil (UUUUUUUUU) at the 3' end of the sgRNA sequence.
- modified sgRNAs can comprise one or more 2'-O-methyl phosphorothioate nucleotides.
- RNAs used in the CRISPR/Cas/Cpfl system can be readily synthesized by chemical means, as illustrated below and described in the art. While chemical synthetic procedures are continually expanding, purifications of such RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more challenging as polynucleotide lengths increase significantly beyond a hundred or so nucleotides.
- HPLC high performance liquid chromatography
- One approach used for generating RNAs of greater length is to produce two or more molecules that are ligated together. Much longer RNAs, such as those encoding a Cas9 or Cpfl endonuclease, are more readily generated enzymatically.
- RNA modifications can be introduced during or after chemical synthesis and/or enzymatic generation of RNAs, e.g., modifications that enhance stability, reduce the likelihood or degree of innate immune response, and/or enhance other attributes, as described in the art.
- indel frequency may be determined using a TIDE analysis, which can be used to identify highly efficient gRNA molecules.
- a highly efficient gRNA yields a gene editing frequency of higher than 80%.
- a gRNA is considered to be highly efficient if it yields a gene editing frequency of at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
- gene disruption may occur by deletion of a genomic sequence using two guide RNAs.
- Methods of using CRISPR-Cas gene editing technology to create a genomic deletion in a cell are known (Bauer D E et al. Vis. Exp. 2015; 95;e52118).
- a gRNA comprises a spacer sequence.
- a spacer sequence is a sequence (e.g., a 20 nucleotide sequence) that defines the target sequence (e.g., a DNA target sequences, such as a genomic target sequence) of a target nucleic acid of interest.
- the spacer sequence is 15 to 30 nucleotides.
- the spacer sequence is 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.
- a spacer sequence is 20 nucleotides.
- the “target sequence” is adjacent to a PAM sequence and is the sequence modified by an RNA-guided nuclease (e.g., Cas9).
- the “target nucleic acid” is a doublestranded molecule: one strand comprises the target sequence and is referred to as the “PAM strand,” and the other complementary strand is referred to as the “non-PAM strand.”
- PAM strand the target sequence
- non-PAM strand complementary strand
- the gRNA spacer sequence hybridizes to the reverse complement of the target sequence, which is located in the non-PAM strand of the target nucleic acid of interest.
- the gRNA spacer sequence is the RNA equivalent of the target sequence.
- the gRNA spacer sequence is 5'-AGAGCAACAGUGCUGUGGCC-3'.
- the spacer of a gRNA interacts with a target nucleic acid of interest in a sequence-specific manner via hybridization (z.e., base pairing).
- the nucleotide sequence of the spacer thus varies depending on the target sequence of the target nucleic acid of interest.
- the spacer sequence is designed to hybridize to a region of the target nucleic acid that is located 5' of a PAM of the Cas9 enzyme used in the system.
- the spacer may perfectly match the target sequence or may have mismatches.
- Each Cas9 enzyme has a particular PAM sequence that it recognizes in a target DNA.
- S. pyogenes recognizes in a target nucleic acid a PAM that comprises the sequence 5'-NRG-3', where R comprises either A or G, where N is any nucleotide and N is immediately 3' of the target nucleic acid sequence targeted by the spacer sequence.
- the target nucleic acid sequence comprises 20 nucleotides. In some embodiments, the target nucleic acid comprises less than 20 nucleotides. In some embodiments, the target nucleic acid comprises more than 20 nucleotides. In some embodiments, the target nucleic acid comprises at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. In some embodiments, the target nucleic acid comprises at most: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. In some embodiments, the target nucleic acid sequence comprises 20 bases immediately 5' of the first nucleotide of the PAM.
- the target nucleic acid comprises the sequence that corresponds to the Ns, wherein N is any nucleotide, and the underlined NRG sequence is the S. pyogenes PAM.
- the gRNAs of the present disclosure are produced by a suitable means available in the art, including but not limited to in vitro transcription (IVT), synthetic and/or chemical synthesis methods, or a combination thereof. Enzymatic (IVT), solid-phase, liquid-phase, combined synthetic methods, small region synthesis, and ligation methods are utilized. In one embodiment, the gRNAs are made using IVT enzymatic synthesis methods. Methods of making polynucleotides by IVT are known in the art and are described in International Application PCT/US2013/30062. Accordingly, the present disclosure also includes polynucleotides, e.g., DNA, constructs and vectors are used to in vitro transcribe a gRNA described herein.
- non-natural modified nucleobases are introduced into polynucleotides, e.g., gRNA, during synthesis or post-synthesis.
- modifications are on intemucleoside linkages, purine or pyrimidine bases, or sugar.
- a modification is introduced at the terminal of a polynucleotide; with chemical synthesis or with a polymerase enzyme. Examples of modified nucleic acids and their synthesis are disclosed in PCT application No. PCT/US2012/058519. Synthesis of modified polynucleotides is also described in Verma and Eckstein, Annual Review of Biochemistry, vol. 76, 99-134 (1998).
- enzymatic or chemical ligation methods are used to conjugate polynucleotides or their regions with different functional moieties, such as targeting or delivery agents, fluorescent labels, liquids, nanoparticles, etc.
- Conjugates of polynucleotides and modified polynucleotides are reviewed in Goodchild, Bioconjugate Chemistry, vol. 1(3), 165-187 (1990).
- nucleic acids e.g., vectors, encoding gRNAs described herein.
- the nucleic acid is a DNA molecule.
- the nucleic acid is an RNA molecule.
- the nucleic acid comprises a nucleotide sequence encoding a crRNA.
- the nucleotide sequence encoding the crRNA comprises a spacer flanked by all or a portion of a repeat sequence from a naturally-occurring CRISPR/Cas system.
- the nucleic acid comprises a nucleotide sequence encoding a tracrRNA.
- the crRNA and the tracrRNA is encoded by two separate nucleic acids. In other embodiments, the crRNA and the tracrRNA is encoded by a single nucleic acid. In some embodiments, the crRNA and the tracrRNA is encoded by opposite strands of a single nucleic acid. In other embodiments, the crRNA and the tracrRNA is encoded by the same strand of a single nucleic acid.
- the gRNAs provided by the disclosure are chemically synthesized by any means described in the art (see e.g., WO/2005/01248). While chemical synthetic procedures are continually expanding, purifications of such RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more challenging as polynucleotide lengths increase significantly beyond a hundred or so nucleotides.
- HPLC high performance liquid chromatography
- One approach used for generating RNAs of greater length is to produce two or more molecules that are ligated together.
- the gRNAs provided by the disclosure are synthesized by enzymatic methods (e.g., in vitro transcription, IVT).
- RNA modifications can be introduced during or after chemical synthesis and/or enzymatic generation of RNAs, e.g., modifications that enhance stability, reduce the likelihood or degree of innate immune response, and/or enhance other attributes, as described in the art.
- more than one guide RNA can be used with a CRISPR/Cas nuclease system.
- Each guide RNA may contain a different targeting sequence, such that the CRISPR/Cas system cleaves more than one target nucleic acid.
- one or more guide RNAs may have the same or differing properties such as activity or stability within the Cas9 RNP complex.
- each guide RNA can be encoded on the same or on different vectors. The promoters used to drive expression of the more than one guide RNA is the same or different.
- the guide RNA may target any sequence of interest via the targeting sequence (e.g., spacer sequence) of the crRNA.
- the degree of complementarity between the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule is about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%.
- the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule is 100% complementary.
- the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain at least one mismatch.
- the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain 1-6 mismatches. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain 5 or 6 mismatches.
- the length of the targeting sequence may depend on the CRISPR/Cas9 system and components used. For example, different Cas9 proteins from different bacterial species have varying optimal targeting sequence lengths. Accordingly, the targeting sequence may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length. In some embodiments, the targeting sequence may comprise 18-24 nucleotides in length. In some embodiments, the targeting sequence may comprise 19-21 nucleotides in length. In some embodiments, the targeting sequence may comprise 20 nucleotides in length.
- a CRISPR/Cas nuclease system includes at least one guide RNA.
- the guide RNA and the Cas protein may form a ribonucleoprotein (RNP), e.g., a CRISPR/Cas complex.
- the guide RNA may guide the Cas protein to a target sequence on a target nucleic acid molecule (e.g., a genomic DNA molecule), where the Cas protein cleaves the target nucleic acid.
- the CRISPR/Cas complex is a Cpfl /guide RNA complex.
- the CRISPR complex is a Type-II CRISPR/Cas9 complex.
- the Cas protein is a Cas9 protein.
- the CRISPR/Cas9 complex is a Cas9/guide RNA complex.
- a gRNA and an RNA-guided nuclease are delivered to a cell separately, either simultaneously or sequentially. In some embodiments, a gRNA and an RNA-guided nuclease are delivered to a cell together. In some embodiments, a gRNA and an RNA-guided nuclease are pre-complexed together to form a ribonucleoprotein (RNP).
- RNP ribonucleoprotein
- RNPs are useful for gene editing, at least because they minimize the risk of promiscuous interactions in a nucleic acid-rich cellular environment and protect the RNA from degradation.
- Methods for forming RNPs are known in the art.
- an RNP containing an RNA-guided nuclease e.g., a Cas nuclease, such as a Cas9 nuclease) and a gRNA targeting a gene of interest is delivered a cell (e.g.: a T cell).
- an RNP is delivered to a T cell by electroporation.
- a “AAVS1 or ROSA26 targeting RNP” refers to a gRNA that targets the AAVS1 or ROSA26 genes pre-complexed with an RNA-guided nuclease.
- a AAVS1 or ROSA26 targeting RNP is delivered to a cell.
- more than one RNP is delivered to a cell.
- more than one RNA is delivered to a cell separately.
- more than one RNP is delivered to the cell simultaneously.
- an RNA-guided nuclease is delivered to a cell in a DNA vector that expresses the RNA-guided nuclease, an RNA that encodes the RNA-guided nuclease, or a protein.
- a gRNA targeting a gene is delivered to a cell as an RNA, or a DNA vector that expresses the gRNA.
- RNA-guided nuclease may be through direct injection or cell transfection using known methods, for example, electroporation or chemical transfection. Other cell transfection methods may be used.
- Delivery of an RNA-guided nuclease, gRNA, and/or an RNP may be through direct injection or cell transfection using known methods, for example, electroporation or chemical transfection. Other cell transfection methods may be used. 6. Multi-Modal or Differential Delivery of Components
- Genome editing systems can be delivered together or separately and simultaneously or nonsimultaneously. Separate and/or asynchronous delivery of genome editing system components may be particularly desirable to provide temporal or spatial control over the function of genome editing systems and to limit certain effects caused by their activity.
- Different or differential modes as used herein refer to modes of delivery that confer different pharmacodynamic or pharmacokinetic properties on the subject component molecule, e.g, a RNA-guided nuclease molecule, gRNA, template nucleic acid, or payload.
- the modes of delivery can result in different tissue distribution, different halflife, or different temporal distribution, e.g, in a selected compartment, tissue, or organ.
- Some modes of delivery e.g., delivery by a nucleic acid vector that persists in a cell, or in progeny of a cell, e.g., by autonomous replication or insertion into cellular nucleic acid, result in more persistent expression of and presence of a component.
- examples include viral, e.g., AAV or lentivirus, delivery.
- the components of a genome editing system can be delivered by modes that differ in terms of resulting halflife or persistent of the delivered component the body, or in a particular compartment, tissue or organ.
- a gRNA can be delivered by such modes.
- the RNA-guided nuclease molecule component can be delivered by a mode which results in less persistence or less exposure to the body or a particular compartment or tissue or organ.
- a first mode of delivery is used to deliver a first component and a second mode of delivery is used to deliver a second component.
- the first mode of delivery confers a first pharmacodynamic or pharmacokinetic property.
- the first pharmacodynamic property can be, e.g., distribution, persistence, or exposure, of the component, or of a nucleic acid that encodes the component, in the body, a compartment, tissue or organ.
- the second mode of delivery confers a second pharmacodynamic or pharmacokinetic property.
- the second pharmacodynamic property can be, e.g., distribution, persistence, or exposure, of the component, or of a nucleic acid that encodes the component, in the body, a compartment, tissue or organ.
- the first pharmacodynamic or pharmacokinetic property e.g., distribution, persistence or exposure, is more limited than the second pharmacodynamic or pharmacokinetic property.
- the first mode of delivery is selected to optimize, e.g., minimize, a pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure.
- the second mode of delivery is selected to optimize, e.g., maximize, a pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure.
- the first mode of delivery comprises the use of a relatively persistent element, e.g., a nucleic acid, e.g., a plasmid or viral vector, e.g., an AAV, adenovirus or lentivirus.
- a relatively persistent element e.g., a nucleic acid, e.g., a plasmid or viral vector, e.g., an AAV, adenovirus or lentivirus.
- a relatively persistent element e.g., a nucleic acid, e.g., a plasmid or viral vector, e.g., an AAV, adenovirus or lentivirus.
- the second mode of delivery comprises a relatively transient element, e.g., an RNA or protein.
- the first component comprises gRNA
- the delivery mode is relatively persistent, e.g., the gRNA is transcribed from a plasmid or viral vector, e.g., an AAV, adenovirus or lentivirus. Transcription of these genes would be of little physiological consequence because the genes do not encode for a protein product, and the gRNAs are incapable of acting in isolation.
- the second component a RNA-guided nuclease molecule, is delivered in a transient manner, for example as mRNA encoding the protein or as protein, ensuring that the full RNA-guided nuclease molecule/gRNA complex is only present and active for a short period of time.
- the components can be delivered in different molecular form or with different delivery vectors that complement one another to enhance safety and tissue specificity.
- differential delivery modes can enhance performance, safety, and/or efficacy, e.g., the likelihood of an eventual off-target modification can be reduced.
- Delivery of immunogenic components, e.g., Cas9 molecules, by less persistent modes can reduce immunogenicity, as peptides from the bacterially-derived Cas enzyme are displayed on the surface of the cell by WIC molecules.
- a two-part delivery system can alleviate these drawbacks.
- a first component e.g., a gRNA is delivered by a first delivery mode that results in a first spatial, e.g., tissue, distribution.
- a second component e.g., a RNA-guided nuclease molecule is delivered by a second delivery mode that results in a second spatial, e.g., tissue, distribution.
- the first mode comprises a first element selected from a liposome, nanoparticle, e.g., polymeric nanoparticle, and a nucleic acid, e.g., viral vector.
- the second mode comprises a second element selected from the group.
- the first mode of delivery comprises a first targeting element, e.g., a cell specific receptor or an antibody, and the second mode of delivery does not include that element.
- the second mode of delivery comprises a second targeting element, e.g., a second cell specific receptor or second antibody.
- RNA-guided nuclease molecule When the RNA-guided nuclease molecule is delivered in a virus delivery vector, a liposome, or polymeric nanoparticle, there is the potential for delivery to and therapeutic activity in multiple tissues, when it may be desirable to only target a single tissue.
- a two-part delivery system can resolve this challenge and enhance tissue specificity. If the gRNA and the RNA-guided nuclease molecule are packaged in separated delivery vehicles with distinct but overlapping tissue tropism, the fully functional complex is only be formed in the tissue that is targeted by both vectors.
- the engineered B cells will be delivered as a therapeutic to a patient in need thereof.
- the engineered B cells will be capable of treating or preventing various diseases or disorders relating to Fabry disease.
- the invention comprises a pharmaceutical composition comprising a population of gene edited B cells comprising at least one therapeutic protein as described herein and a pharmaceutically acceptable excipient.
- the pharmaceutical composition further comprises an additional active agent.
- pharmaceutical compositions according to the invention are administered to a subject in a dosage sufficient to achieve a therapeutic effect.
- therapeutic effect or action includes an effect or action of a pharmaceutical composition of the invention intended to cure, mitigate, treat, or prevent, or stabilize the progression of, a disease, disorder or condition, or affect the structure or any function of the body of a subject.
- appropriate doses and dosing schedules may be vary according to the subject and the intended therapeutic effect, and may dependent upon such factors as, for example, the disease, disorder or condition being treated, and the general health, age, body weight, sex, or relevant genetic makeup of the subject. Such appropriate dose levels and dosing schedules can be determined by the healthcare provider as needed. Additionally, multiple doses of engineered B cells can be provided in accordance with the invention.
- the expanded population of engineered B cells are autologous B cells.
- the engineered B cells are allogeneic B cells.
- the engineered B cells are heterologous B cells.
- the engineered B cells of the present application are prepared by in vivo transfection or in vivo transduction. In other embodiments, the engineered cells are prepared ex vivo by transfection or transduction.
- a subject or “patient” means an individual.
- a subject is a mammal such as a human.
- a subject can be a nonhuman primate.
- Non-human primates include marmosets, monkeys, chimpanzees, gorillas, orangutans, and gibbons, to name a few.
- subject also includes domesticated animals, such as cats, dogs, etc., livestock (e.g., llama, horses, cows), wild animals (e.g., deer, elk, moose, etc.,), laboratory animals (e.g., mouse, rabbit, rat, gerbil, guinea pig, etc.) and avian species (e.g, chickens, turkeys, ducks, etc.).
- livestock e.g., llama, horses, cows
- wild animals e.g., deer, elk, moose, etc.
- laboratory animals e.g., mouse, rabbit, rat, gerbil, guinea pig, etc.
- avian species e.g, chickens, turkeys, ducks, etc.
- the subject is a human subject. More preferably, the subject is a human patient.
- the composition comprising gene edited B cells can be administered with an anti-inflammatory agent.
- Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate.
- steroids and glucocorticoids including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triam
- Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylates.
- Exemplary analgesics include acetaminophen, oxycodone, tramadol of proporxyphene hydrochloride.
- Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone.
- Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as the TNF antagonists, (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®) and infliximab (REMICADE®)), chemokine inhibitors and adhesion molecule inhibitors.
- TNF antagonists e.g., etanercept (ENBREL®), adalimumab (HUMIRA®) and infliximab (REMICADE®)
- chemokine inhibitors esion molecule inhibitors.
- adhesion molecule inhibitors include monoclonal antibodies as well as recombinant forms of molecules.
- Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular) and minocycline.
- compositions described herein are administered in conjunction with a cytokine.
- cytokine as used herein is meant to refer to proteins released by one cell population that act on another cell as intercellular mediators. Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones.
- growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors (NGFs) such as NGF-beta; platelet-growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoin
- FSH follicle
- the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of the native sequence cytokines.
- the method further comprises administering at least a second therapy to the subject.
- the method further comprises a second therapy for the treatment of Fabry disease.
- the therapy is an oral chaperone therapy.
- the second therapy is Galafold® (migalastat).
- the second therapy is enzyme replacement therapy.
- the enzyme replacement therapy is an infusion of recombinant a-GAL.
- the second therapy is a substrate reduction therapy.
- the second therapy is a therapy to reduce how much Gb3 is made by blocking other enzymes necessary for its production.
- Human B cells were isolated from healthy donors using magnetic beads and activated using CD40 ligand and IL21.
- B cells were engineered using an rAAV6 encoding a- GAL protein and either an AAVS1 specific or ROSA26 specific RNP. See FIG. 1 A (AAVS1) and FIG. IB (ROSA26).
- B cells were edited to express the WT human a-GAL protein (1137, SEQ ID No. 9), the a-GAL protein with a carboxy -terminal LL truncation of the human a-GAL protein (1138, SEQ ID No.
- B cells were only transduced with rAAV6 in absence of RNP electroporation to determine the level of a-GAL activity from AAV6 episomal expression in B cells (see, e.g., FIG. 2A-B “Episomal”).
- AAVS1, ROSA26 sgRNAs and CRISPR engineering A chemically modified sgRNA oligomer targeting AA VS1 or ROSA26 was manufactured by IDT (Integrated DNA Technologies, Coralville, Iowa, USA). Recombinant S. pyogenes Cas9 enzyme was purchased from IDT (Integrated DNA Technologies, Coralville, Iowa, USA). Cas9 was incubated with sgRNA at a molar ratio of 1 : 1.6 at room temperature for 10 minutes prior to mixing with B cells. Engineering of primary human B cells was carried out using an AMAXATM 4D-NUCLEOF ACTORTM in P3 nucleofection solution with program CM-137 (Lonza, Basel, Switzerland).
- RNP 100 pmol RNP was used for electroporation with 1 million activated human B cells in 20 pl volume. Immediately after electroporation, B cells were transduced with rAAV6 donor at a multiplicity of infection (MOI) of 100,000 viral genomes (vg)/pl to maximize efficiency of transduction.
- MOI multiplicity of infection
- a-GAL activity Activity of secreted a-GAL forms from gene edited human B cells differentiated to PBs or PCs was then measured. Supernatants from edited human B cells were collected at 6, 9 and 12 days after gene editing. A-GAL specific activity was measured using the Alpha Galactosidase Activity Assay kit according to manufacturer’s instruction (Abeam, ab239716).
- Example 2 A-GAL Activity in Differentiated Plasma Blasts and Plasma Cells
- human B cells isolated from peripheral blood were edited at the AAVS1 locus to again express the WT ha-GAL protein (SEQ ID No. 1), the a-GAL protein with a carboxy-terminal LL truncation of the ha-GAL protein (SEQ ID No. 2), or a a-GAL protein with both the carboxy-terminal LL truncation and the addition of a heavy chain (SEQ ID No. 3) or light chain (SEQ ID No. 4) signal peptide.
- B cells were only transduced with rAAV6 vectors, without RNP nucleofection (“episomal”). The B cells were next differentiated toward either PBs or PCs, and a-GAL activity was measured using the protocol described in Example 1.
- a-GAL expression was measured in edited cells under modified conditions.
- the IFNa2b concentration was increased from 1.5 ng/ml to 15 ng/ml.
- Cellular density was maintained at 0.6 M/mL and cells were expanded for nine days instead of seven.
- edited peripheral blood cells differentiated toward PBs and PCs showed comparable secretion of active a-GAL when edited with a truncated version of a-GAL (“Edited GLA (1140)").
- Construct 1482 is the original cDNA sequence (SEQ ID NO. 4). Of the five codon optimized constructs (1588 (SEQ ID No. 26), 1598 (SEQ ID No. 27), 1599 (SEQ ID No. 28), 1600 (SEQ ID No. 29) and 1606 (SEQ ID No. 25)), construct 1606 and 1599 showed enhanced a- GAL activity in B cells/PB cells when compared to activated B cells and compared to construct 1482.
- Example 6 Restoration of Intracellular a-GAL levels in Fabry Patient Fibroblasts
- Fabry’s disease is characterized by a deficiency in the body’s ability to produce a- GAL enzyme.
- M6P-R M6P receptors
- FIG. 7A fibroblasts from patients with Fabry Disease were isolated and grown as primary cell culture. Fibroblasts were then co-cultured with the supernatant from HEK 293 expressing/secreting either WT a- GAL or a-GAL with both the carboxy -terminal LL truncation and the addition of a heavy chain signal peptide (SEQ ID No. 3).
- a-GAL activity and expression of ha-GAL protein from edited B cells was examined in vivo. Mice were injected with 30 x 10 6 of either un-edited or edited, activated B cells along with 3 x 10 6 CD4 + T cells. Engraftment and survival of human B cells in NSG is poor given the lack of additional human immune components and cytokines. CD4 + T cells play an important role in survival and function of B cells. It has been demonstrated that the co-transfer of CD4 + T cells supports survival and function of human B cells in NSG mice. See, e.g., Ishikawa, Y., et al., 2014, Eur. J. Immunol., 44:3453-3463; Luo B. et al., 2020, Cell Death and Disease, 11 :973.
- a-GAL activity (FIG. 9A) was measured over 102 days and protein expression (FIG. 9B) was measured over the course of 22 days.
- a-GAL activity was measured from isolated plasma as described in Example 1 above.
- a-GAL specific activity in plasma of NSG mice was measured using the Alpha Galactosidase Activity Assay kit according to manufacturer’s instruction (Abeam, ab239716).
- a-GAL protein concentration was assessed using Human GLA/ Alpha Galactosidase (Sandwich ELISA) (LS Bio, LS-F 10765).
- Example 8 Assessment of sgRNA efficiency and specificity and effect of codon optimization on a-GAL expression.
- Described herein is a highly efficient, specific and safe gene editing strategy targeting an AAV1 locus.
- Healthy donor B cells are isolated from peripheral blood and engineered to expresses a-GAL, using CRISPR/Cas-based genome editing using the methods described above.
- Engineered B cells were expanded and cultured using the experimental methods described above and as set forth in FIG. 13A and FIG. 13B.
- FIG. 11 A SEQ ID NO. 11
- FIG. 1 IB SEQ ID NO. 25
- B Cell phenotype was assessed in engineered B cells after in vitro conditioning.
- a-GAL activity protein from edited B cells was examined in vivo. Mice were injected with 30 x 10 6 of either un-edited or edited, activated B cells along with 3 x 10 6 CD4 + T cells as described in Example 7 above. Human B cells gene edited and cultured as described in Example 1 were harvested at D6 after editing and washed in PBS. Subsequently, B cells were resuspended at 30 xlO 6 cells/300 ul and transferred into NSG mice via intravenous tail vein injection. Five days prior B cells transfer total 3 xlO 6 CD4 + T cells were transferred into the NSG mice using the same procedure. Plasma was isolated from NGS mice as described in Example 7 above and a-GAL activity was measured from isolated plasma as described in Example 1 above.
- Engineered B Cells engrafted in NSG mice produced sustained, supraphysiologic a-GAL plasma levels (FIG. 15).
- Luciferase imaging showed early engraftment of engineered cells in bone marrow (FIG. 16A).
- Example 11 Reduction in GvHD symptoms by infusion of memory CD4+ cells.
- NSG mice [0192] B cell engraftment in NSG mice is extremely poor in the absence of support from additional human immune components or cytokines (Luo 2020; Cheng 2022). NSG mice humanized via adoptive transfer of autologous total human CD4+ T cells prior or together with human B cells dramatically increased the ability of B cells to engraft and survive in vivo (Levy 2016).
- CD4+ T cells were to reduce the incidence and delay the onset of GvHD.
- a-GAL activity and expression of ha-GAL protein from edited B cells was examined in vivo.
- Mice were injected with 25 x 10 6 of either un-edited or edited, activated B cells along with 3 x 10 6 CD4 + T cells as described in Example 7 above.
- Human B cells gene edited and cultured as described in Example 1 were harvested at D6 after editing and washed in PBS. Subsequently, B cells were resuspended at 25 xlO 6 cells/300 pl and transferred into NSG mice via intravenous tail vein injection. Five days prior B cells transfer total 3 xlO 6 CD4 + T cells were transferred into the NSG mice using the same procedure.
- Plasma was isolated from NGS mice as described in Example 7 above and a-GAL activity was measured from isolated plasma as described in Example 1 above.
- Circulating a-GAL levels were monitored every 7-10 days via an enzymatic activity assay on plasma samples.
- Engineered B Cells engrafted in NSG mice produced sustained, supraphysiologic a-GAL plasma levels (FIG. 18).
- NSG mice were humanized as described in FIG 18 were assessed for bodyweight and GVHD symptoms (e.g., hair loss, redness ears/extremities, hunched posture).
- mice infused with Memory CD4+ T cells together with edited B cells (1140, SEQ ID NO. 11) (FIG. 19B) showed no difference in body weight or GVHD symptoms as compared to saline controls.
- mice infused with Total CD4+ T cells together with edited B cells (1140, SEQ ID NO. 11) showed reduction in body weight and/or exhibited GVHD symptoms. (FIG. 19C).
- human B cells isolated from peripheral blood of a Fabry patient activated and were edited at the AAVS1 locus to again express construct 1606 (SEQ ID NO. 25).
- B cells were isolated from Fabry patient’s PBMCs via CD19+ positive selection, and activated for 3 days in media containing CD40L, CpG, IL-2, IL- 10, IL- 15 and IL-21.
- Gene editing was performed on day 3 after isolation using Cas9/sgRNA delivered via electroporation followed by transduction via AAV6 GLA vector (1606, SEQ ID No. 25).
- Engineered B cells were expanded in the same media describe above for additional 7 days.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Diabetes (AREA)
- Plant Pathology (AREA)
- Epidemiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Disclosed herein are novel compositions and methods for the treatment of Fabry disease, including therapeutic proteins with enhanced α-galactosidase (α-GAL) activity and engineered B cells edited to express modified therapeutic proteins for the treatment of Fabry disease. Also disclosed, herein, are methods for manufacturing the disclosed therapeutic proteins and engineered B cells, and their methods for use in the treatment of Fabry disese.
Description
COMPOSITIONS AND METHODS FOR TREATMENT OF FABRY DISEASE
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit and priority of U.S. Provisional Application No. 63/400,664, filed on August 24, 2022, and U.S. Provisional Application No. 63/484,668, filed on February 13, 2023, the contents of each of which are incorporated herein by reference in their entireties.
BACKGROUND OF THE INVENTION
[0002] Fabry disease is an incurable, progressive lysosomal storage disease characterized by mutations in the GLA gene, which results in the absence or decreased function of the encoded alpha-galactosidase A (a-GAL) enzyme, leading to the accumulation of Globotriaosylceramide (Gb3) throughout the body. The classic presentation of the disease begins with pain in the first to second decade of life followed by progressive renal, cardiac, neurologic, GI and dermal involvement. Classically affected patients typically succumb to the disease in their 50s.
[0003] Oral chaperone therapy is available to a subset of patients who have amenable GLA-mutations; however, the majority of patients are treated with enzyme replacement therapy (ERT): a bi-weekly infusion of recombinant a-GAL. a-GAL ERT therapy is a lifelong commitment typically requiring infusions that can last for several hours, frequently cause acute infusion reactions, results in highly variable plasma a-GAL concentrations due to short a-GAL half-life, and places a large burden on financial and other resources within the health care system. Due to these challenges, long-term compliance is difficult. A need for novel and more universal treatment of Fabry disease is remains.
SUMMARY OF THE INVENTION
[0004] Disclosed herein are novel compositions and methods for the treatment of Fabry disease.
[0005] In various embodiments, the invention relates to a therapeutic protein comprising a modified human a-GAL protein, wherein the modified human a-GAL protein has been modified by, a deletion of two carboxy terminal leucine residues of a wild-type human a-
GAL protein; and a replacement of a signal peptide of said wild-type human a-GAL protein with an immunoglobulin(Ig) signal peptide.
[0006] In various embodiments, the signal peptide is a heavy chain Ig signal peptide. In various embodiments, the signal peptide is a light chain Ig signal peptide. In various embodiments, the therapeutic protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments, the therapeutic protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments, the therapeutic protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments, the therapeutic protein comprises an amino acid sequence that is selected from: SEQ ID Nos: 2-4. In various embodiments, the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the modified human a- GAL protein is encoded by a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the modified human a-GAL protein is encoded by a nucleic acid sequence is selected from: SEQ ID Nos: 6-8, 16-17 or 20-21.
[0007] In various embodiments the invention relates to an engineered human B cell comprising a therapeutic protein, wherein the therapeutic protein comprises a modified human GLA wherein the modified human a-GAL protein has been modified by, a deletion of two carboxy terminal leucine residues of a wild-type human a-GAL protein; and a replacement of a signal peptide of said wild-type human a-GAL protein with an immunoglobulin(Ig) signal peptide.
[0008] In various embodiments, the engineered B cell further comprises a nucleic acid sequence encoding said therapeutic protein, and wherein said nucleic acid sequence has been inserted into either the AAVS1 or the human ROSA26 locus. In various embodiments, the signal peptide is a heavy chain Ig signal peptide. In various embodiments, the signal peptide is a light chain Ig signal peptide. In various embodiments, the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 85% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments, the modified human a-
GAL protein is encoded by a nucleic acid sequence that is at least 90% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments, the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 95% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments, the modified human a-GAL protein is encoded by a nucleic acid sequence that is selected from: SEQ ID Nos: 2-4. In various embodiments, the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the modified human a- GAL protein is encoded by a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the modified human a-GAL protein is encoded by a nucleic acid sequence that is selected from: SEQ ID Nos: 6-8, 16-17 or 20-21.
[0009] In various embodiments, the gene has been inserted into the human AAVS1 locus. In various embodiments, the gene has been inserted into the human ROSA26 locus. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells, wherein at least 20% of the population of cells express said therapeutic protein. In various embodiments said B cell is CD20+. In various embodiments said B cell is CD20-. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells, wherein a majority of said cells are CD20+. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells, wherein more than 80 percent of said cells are CD20-. In various embodiments, said B cell is a plasmablast. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells, wherein a majority of said cells are plasmablasts. In various embodiments, said cell is a plasma cell. In various embodiments, a population of cells comprising a plurality of engineered B cells, wherein a majority of said cells are plasma cells.
[0010] In various embodiments, the invention relates to a method of producing an engineered B cell expressing a therapeutic protein, the method comprising delivering to a
human B cell: an RNA-guided nuclease; a gRNA targeting a locus on the human genome; and a construct comprising a nucleic acid sequence encoding a therapeutic protein, wherein the therapeutic protein comprises a human a-GAL protein.
[0011] In various embodiments, the RNA-guided nuclease and gRNA are delivered to the B cell as an RNP. In various embodiments, the RNA-guided nuclease and gRNA are delivered to the B cell as a nanoparticle. In various embodiments, the RNA-guided nuclease and gRNA are delivered to the B cell via electroporation. In various embodiments, the construct delivered to the B cell using a viral vector. In various embodiments, the construct delivered to the B cell as double stranded DNA. In various embodiments, the RNA-guided nuclease comprises the amino acid sequence of SEQ ID No. 14 or SEQ ID No. 24. In various embodiments, the RNA-guided nuclease comprises the amino acid sequence of SEQ ID NO. 15. In various embodiments, the targeting construct comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 9-13 18-19 and 25-29.
[0012] In various embodiments, said engineered B cell is CD20-. In various embodiments, said engineered B cell is CD20+. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells, wherein a majority of said cells are CD20-. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells, wherein more than 80 percent of said cells are CD20+. In various embodiments, said engineered B cell is a plasmablast. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells, wherein a majority of said cells are plasmablasts. In various embodiments, said engineered B cell is a plasma cells. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells, wherein a majority of said cells are plasma cells.
[0013] In various embodiments, the invention relates to a method of producing an engineered B cell expressing a therapeutic protein, the method comprising delivering to a human B cell: a RNA-guided nuclease; a gRNA targeting the AAVS1 gene, wherein the gRNA comprises the nucleic acid sequence of SEQ ID NO. 14, 15 or 24; and a construct comprising a nucleic acid sequence encoding a therapeutic protein and a left and right homology arm; wherein the construct comprises a nucleic acid sequence selected from the
group consisting of SEQ ID NOs. 9-13 and 18-19, wherein said engineered B cell expresses said therapeutic protein.
[0014] In various embodiments, the invention relates to a method of treating a patient in need thereof comprising administering to said patient an engineered human B cell, wherein the engineered human B cell has been edited to express a therapeutic protein encoding a human a-GAL protein. In various embodiments, the therapeutic protein comprises a modified human a-GAL protein, wherein the two carboxy terminal leucine residues of the human a-GAL protein have been deleted; and the GLA signal peptide has been replaced by an immunoglobulin (Ig) signal peptide. In various embodiments, the signal peptide is a heavy chain Ig signal peptide. In various embodiments, the signal peptide is a light chain Ig signal peptide. In various embodiments, the therapeutic protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments, the therapeutic protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments, the therapeutic protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments, the therapeutic protein comprises an amino acid sequence that is selected from: SEQ ID Nos: 2-4. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20- 21.
[0015] In various embodiments, the gene encoding the therapeutic protein has been inserted into the human AAVS1 locus. In various embodiments, the gene encoding the therapeutic protein has been inserted into the human ROSA26 locus. In various embodiments, at least 20% of the population of cells express said therapeutic protein. In various embodiments, said population of cells comprise cells that are CD20+. In various embodiments, said population of cells comprise cells that are CD20+. In various
embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells according to any one of claims 54-64, wherein a majority of said cells are CD20+. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells according to any one of claims 54-64, wherein more than 80 percent of said cells are CD20-. In various embodiments, said engineered B cell is a plasmablast. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells, wherein a majority of said cells are plasmablasts. In various embodiments, said engineered B cell is a plasma cells. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells, wherein a majority of said cells are plasma cells.
[0016] In various embodiments, the invention relates to, a method of treating a patient in need thereof comprising administering to said patient an engineered human B cell, wherein said human B cell comprises a therapeutic protein, encoded by a gene, wherein the therapeutic protein comprises a modified human a-GAL protein, wherein the two carboxy terminal leucine residues of the human GLA protein have been deleted; and the GLA signal peptide has been replaced by an immunoglobulin (Ig) signal peptide.
[0017] In various embodiments, the nucleic acid sequence encoding said therapeutic protein has been inserted into either the AAVS1 or the human ROSA26 locus. In various embodiments, the signal peptide is a heavy chain Ig signal peptide. In various embodiments, the signal peptide is a light chain Ig signal peptide. In various embodiments, the therapeutic protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments, the therapeutic protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments, the therapeutic protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments, the therapeutic protein comprises an amino acid sequence that is selected from: SEQ ID Nos: 2-4. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence that is
at least 95% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21.
[0018] In various embodiments, the gene has been inserted into the human AAVS1 locus. In various embodiments, the gene has been inserted into the human ROSA26 locus. In various embodiments, at least 20% of the population of engineered B cells, express said therapeutic protein. In various embodiments, said population of cells comprise cells that are CD20+. In various embodiments, said population of cells comprise cells that are CD20-. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells wherein a majority of said cells are CD20+. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells, wherein more than 80 percent of said cells are CD20-. In various embodiments, said engineered B cell is a plasmablast. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells, wherein a majority of said cells are plasmablasts. In various embodiments, said engineered B cell is a plasma cells. In various embodiments, the invention relates to a population of cells comprising a plurality of engineered B cells, wherein a majority of said cells are plasma cells.
[0019] In various embodiments, the invention relates to a method of treating a patient in need thereof comprising administering to said patient a therapeutic protein comprising a modified human a-GAL protein, wherein the two carboxy terminal leucine residues have been deleted; and the GLA signal peptide has been replaced by an immunoglobulin (Ig) signal peptide.
[0020] In various embodiments, the signal peptide is a heavy chain Ig signal peptide. In various embodiments, the signal peptide is a light chain Ig signal peptide. In various embodiments, the therapeutic protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments, the therapeutic protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments, the therapeutic protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. In various embodiments the therapeutic protein comprises an amino acid sequence that is selected from:
SEQ ID Nos: 2-4. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20- 21.
BRIEF DESCRIPTION OF THE FIGURES
[0021] FIG. 1 shows a schematic of two insertion sites for targeted integration as per certain embodiments of the present invention. FIG. 1 A shows insertion of the GLA gene at the human AAVS1 locus on chromosome 19. The insert size of this targeting construct is approximately 3700 bp. FIG. IB shows insertion of the GLA gene at the human ROSA26 locus on chromosome 3.
[0022] FIG. 2 shows the a-GAL activity of human B cells gene edited using Cas9-AAV6 mediated integration to express a version of the a-GAL protein (1137 = wild type (WT) GLA (SEQ ID No. 9)); 1138 = GLA with a carboxy -terminal two leucine (LL) truncation (SEQ ID No. 10); 1139 = GLA with LL truncation and an immunoglobulin kappa chain (variable 3-5) signal peptide (SEQ ID No. 13); 1140 = GLA with LL truncation and heavy chain signal peptide(SEQ ID No. 11)). FIG 2A shows a-GAL activity after insertion at the AAVS1 locus. FIG. 2B shows a-GAL activity after insertion at the ROSA26 locus. Under the culture conditions used, peripheral blood B cells become activated and some differentiate towards plasmablasts (PB) and plasma cells (PC). When compared with episomal AAV expression, peripheral blood B cells edited to express human GLA showed significantly enhanced a-GAL activity, indicating integration.
[0023] FIG. 3 shows a-GAL activity in activated B-cells and peripheral blood cells differentiated toward PBs and PCs that have been edited to express various versions of the a- GAL protein (1137 = wild type (WT) GLA (SEQ ID No. 9); 1138 = GLA with a carboxyterminal two leucine (LL) truncation (SEQ ID No. 10); 1140 = GLA with LL truncation and heavy chain signal peptide (SEQ ID No. 11); 1159 = GLA with LL truncation and light chain signal peptide).
[0024] FIG. 4 shows a-GAL activity in peripheral blood cells differentiated toward PBs from five different healthy donors (Donors A-E) that have been edited to express two versions of the a-GAL protein (Donor A, 1137 = Edited Wildtype GLA (SEQ ID No. 9); Donors A-E, 1140 = Edited GLA with LL truncation and heavy chain signal peptide (SEQ ID No. 11)).
[0025] FIG. 5 shows a-GAL activity in activated B cells and peripheral blood cells differentiated toward PBs and PCs. Human B cells were activated for 3 days in a media containing CD40L, CpG, IL2, IL 10, IL 15 and IL21. Gene editing (1140 = GLA with LL truncation and heavy chain signal peptide (SEQ ID No. 11)) was performed at day 3 after isolation and cells were expanded in the same media for an additional 6 days. Subsequently, expanded B cells were culture in a media containing IL2, IL6, IL10, IL15, IL21 for 3 days, to obtain a mixture of plasmablasts and B cells. To promote PCs differentiation, cells were cultured for 3 days in a media supplemented with IL6, IL15, IL21 and IFNa2b. For this experiment, the IFNa2b concentration was 15 ng/ml and cellular density was maintained at 0.6 M/mL. Under these conditions, a-GAL activity was comparable between edited peripheral blood cells differentiated toward PBs and PCs when compared with edited activated B cells and un-edited, mock and AAV transduced cells.
[0026] FIG. 6 shows a-GAL activity in edited human peripheral blood cells differentiated toward PBs. When compared with episomal (AAV) expression, B cells edited to express human GLA (labeled in FIG. 6 as “Edited” followed by the construct number) showed significantly enhanced a-GAL activity. Codon optimized constructs 1509 (SEQ ID No. 18) and 1510 (SEQ ID No. 19) showed significantly enhanced a-GAL activity over other human a-GAL expressing constructs (1501= GLA with 4 amino acids truncation at the C-terminal and heavy chain signal peptide; 1509 (SEQ ID No. 18), 1510 (SEQ ID No. 19), 1511 (SEQ ID No. 23) = 3 different codon optimizations of GLA sequence with LL truncation and heavy chain signal peptide).
[0027] FIG. 7A shows the mechanism by which a-GAL enters cells. Phosphorylated a- GAL binds to M6P receptors (M6P-R) and is internalized in the lysosome. FIG. 7B shows restoration of intracellular a-GAL levels in fibroblasts derived from Fabry patients. Fibroblasts cultured with concentrated supernatant from 293 cells secreting either WT (1002,
SEQ ID No. 5) a-GAL or truncated a-GAL (1067, SEQ ID No. 7) showed increased a-GAL activity when compared with both WT and Fabry fibroblasts alone.
[0028] FIG. 8 shows expression of a-GAL protein from edited B cells in vivo. NSG mice were humanized via adoptive transfer of 3xl06 total CD4+ T cells. Five days post CD4+ T cells transfer, 30xl06 activated edited B cells (1140 = GLA with LL truncation and heavy chain signal peptide (SEQ ID No. 11)) were infused into the humanized mice. FIG. 8 A shows a-GAL enzyme activity measured from plasma samples collected 3 days after B cells transfer and every 10 days up to 100 days. FIG. 8B shows a-GAL protein expression using an ELISA from plasma taken from the same NSG mice on days 3, 10 and 20.
[0029] FIG. 9 shows gene editing efficiency (FIG. 9A) and on-target GLA integration (FIG. 9B in peripheral blood human B cells gene edited using a Ribonucleoproteins (RNP) targeting an AAV1 locus delivered into B cells via nucleofection and GLA cDNA via recombinant AAV6 (rAAV6) transduction. Engineered B cells were cultured using the methods described below and genomic DNA was extracted to measure the efficiency of sitespecific mutations (FIG. 9A) and targeted integration frequency, using digital PCR assays (FIG. 9B) (n=3 health donors).
[0030] FIG. 10 shows that optimized single guide RNA mediate efficient on-target gene editing with no off-target events. RNPs targeting an AAV1 locus were delivered into B cells via nucleofection and genomic DNA was extracted to measure efficiency of site-specific mutations using a digital PCR assay. Efficiency was measured using initially designed sgRNA (FIG. 10 A, n=4, SEQ ID No. 14 GGGGCCACTAGGGACAGGAT) and optimized sgRNA (FIG. 10B, n= 3, SEQ ID No. 24, CCTCTAAGGTTTGCTTACGA).
[0031] FIG. 11 shows the effect of codon optimization on a-GAL activity in vitro. Human B cells were edited using various GLA-cDNA sequences (FIG. 11 A (SEQ ID No. 11) and FIG. 1 IB (SEQ ID No. 25)) and cultured for 12 days. At 9 and 12 days, cell culture supernatants were collected, and cell counts and viability were assessed. Functional, secreted a-GAL was quantified using an activity assay and results were normalized to cell counts.
[0032] FIG. 12 shows a guide-SEQ analysis of genomic DNA isolated from Human B cells. Guide-SEQ analysis of genomic DNA isolated from human B cells treated with safe
harbor specific RNPs. On-target/off-target sites were identified by incorporation of doublestranded oligodeoxynucleotides (dsODNs).
[0033] FIG. 13 A shows an overview of various ex vivo B cell engineering and culture procedures. FIG. 13B shows the kinetics of unedited B cell numbers monitored every 2 days (n= 4 healthy donors).
[0034] FIG. 14 shows B cell lineages after in vitro conditioning. FIG. 14A shows various cell populations, memory B cells (CD27+IgD-), plasmablasts (PBs) (CD27+CD38+ CD20-), and plasma cells (PCs) (CD27+CD38+ CD20- CD 138+) populations monitored using Flow cytometry. FIG. 14B shows PrimeFlow analysis of GLA transcripts in B cell populations.
[0035] FIG. 15 shows sustained, supraphysiologic a-GAL plasma levels in vivo. Human B cells were engineered and cultured as described. GLA-edited B cells were adoptively transferred into NSG mice pre-conditioned with adoptive transfer of CD4+ T cells prior to adoptive transfer of GLA-edited B cells. Circulating a-GAL levels were monitored every 7- 10 days via an enzymatic activity assay on plasma samples (n= 2 healthy donors).
[0036] FIG. 16 shows adoptively transferred genetically engineered B cells engrafted in mice in vivo. Engineered human B cells were adoptively transferred into CD4+ T cell humanized NSG mice. Representative luciferase image shows early engraftment of the engineered cells primarily in the bone marrow (FIG. 16A). Engrafted B cells were isolated from multiple murine tissues and the cell phenotype was assessed using Flow cytometry and relative proportions plotted (n=4 mice) (FIG. 16B).
[0037] FIG. 17 shows characterization of the effect of editing on patient-derived engineered B cells from Fabry patient’s PBMCs. B cells were isolated from Fabry patient’s PBMCs via CD 19+ positive selection, and activated for 3 days in media containing CD40L, CpG, IL-2, IL- 10, IL- 15 and IL-21. Gene editing was performed on day 3 of culture using Cas9/sgRNA delivered via electroporation and by transduction via AAV6 of active enzyme GLA (1606) followed by an additional 7 days of culture. Engineered B cells were expanded in the same media described above for an additional 7 days. FIG. 17A shows that the Fabry edited B cells were capable of expansion and growth. FIG. 17B shows that the Fabry B cell had successful integration. FIG. 17C shows that GLA-Edited Fabry cells showed increased secretion of a-GAL active enzyme when compared to non-edited controls.
[0038] FIG. 18 shows systemic levels of a-GAL increase over time in both total CD4+ and memory CD4+ T cells mouse models. NSG mice were humanized via adoptive transfer of either total human CD4+ cells or memory human CD4+ cells together with edited B cells (1140, SEQ ID No. 11) and a-GAL activity was assessed. Peripheral blood B cells edited to express human GLA showed significantly enhanced a-GAL activity in both mouse models (Memory CD4+ and Total CD4+) when compared to mice injected with saline.
[0039] FIG. 19 shows memory CD4+ T cells mouse model does not show any symptoms of GvHD. NSG mice were humanized as described in FIG 18 were assessed for bodyweight and GVHD symptoms e.g., hair loss, redness ears/extremities, hunched posture). Compared with saline treated mice (FIG. 19A), mice infused with Memory CD4+ T cells together with edited B cells (1140, SEQ ID No. 11) (FIG. 19B) showed no difference in body weight or GVHD symptoms. In contrast, mice infused with Total CD4+ T cells together with edited B cells (1140, SEQ ID No. 11) showed reduction in body weight and/or exhibited GVHD symptoms (FIG. 19C).
[0040] FIG. 20 shows a-GAL activity in edited human PBs using various codon optimized constructs in activated B cells and peripheral blood cells differentiated toward PBs in Donor 1 (FIG. 20A) and Donor 2 (FIG. 20B). When compared with episomal (AAV) expression, B cells edited to express human GLA (“Edited”) showed significantly enhanced a-GAL activity. Construct 1482 is the original cDNA sequence (1140, SEQ ID No. 11). Of the five codon optimized constructs (1588, 1598, 1599, 1600 and 1606), construct 1606 and 1599 showed enhanced a-GAL activity in PB cells when compared to activated B cells and compared to construct 1482.
DETAILED DESCRIPTION
[0041] Disclosed herein are novel methods for the treatment of Fabry disease. Various embodiments disclosed herein provide for autologous human B-cells that have been gene- edited to express a functional a-GAL protein and will provide long-term stable expression of the enzyme in vivo. The advantages of such a cellular therapy include: (i) decreased frequency of infusion (e.g., every 12 months instead of every two weeks); (ii) decreased rate and severity of acute infusion reactions; (iii) improved steady state expression of enzyme which may reduce the risk of immunogenicity and enable better clinical outcome; (iv) potential improved patient compliance and therefore better clinical outcome.
I. Definitions
[0042] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose. As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:
[0043] In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.
[0044] The term “polynucleotide”, “nucleotide”, or “nucleic acid” includes both singlestranded and double-stranded nucleotide polymers. The nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2’, 3 ’-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphoro-diselenoate, phosphoro-anilothioate, phoshoraniladate and phosphoroamidate.
[0045] The term “oligonucleotide” refers to a polynucleotide comprising 200 or fewer nucleotides. Oligonucleotides can be single stranded or double stranded, e.g., for use in the construction of a mutant gene. Oligonucleotides can be sense or antisense oligonucleotides. An oligonucleotide can include a label, including a radiolabel, a fluorescent label, a hapten or an antigenic label, for detection assays. Oligonucleotides can be used, for example, as PCR primers, cloning primers or hybridization probes.
[0046] The term “control sequence” refers to a polynucleotide sequence that can affect the expression and processing of coding sequences to which it is ligated. The nature of such control sequences can depend upon the host organism. In particular embodiments, control sequences for prokaryotes can include a promoter, a ribosomal binding site, and a transcription termination sequence. For example, control sequences for eukaryotes can include promoters comprising one or a plurality of recognition sites for transcription factors,
transcription enhancer sequences, and transcription termination sequence. Control sequences can include leader sequences (signal peptides) and/or fusion partner sequences.
[0047] As used herein, “operably linked” means that the components to which the term is applied are in a relationship that allows them to carry out their inherent functions under suitable conditions.
[0048] The term “vector” means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage or virus) used to transfer protein coding information into a host cell. The term “expression vector” or “expression construct” refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control (in conjunction with the host cell) expression of one or more heterologous coding regions operatively linked thereto. An expression construct can include, but is not limited to, sequences that affect or control transcription, translation, and, if introns are present, affect RNA splicing of a coding region operably linked thereto.
[0049] The term “host cell” refers to a cell that has been transformed, or is capable of being transformed, with a nucleic acid sequence and thereby expresses a gene of interest. The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent cell, so long as the gene of interest is present.
[0050] The term “transformation” refers to a change in a cell’s genetic characteristics, and a cell has been transformed when it has been modified to contain new DNA or RNA. For example, a cell is transformed where it is genetically modified from its native state by introducing new genetic material via transfection, transduction, or other genetic engineering techniques. Following transfection or transduction, the transforming DNA can recombine with that of the cell by physically integrating into a chromosome of the cell, or can be maintained transiently as an episomal element without being replicated, or can replicate independently as a plasmid. A cell is considered to have been “stably transformed” when the transforming DNA is replicated with the division of the cell.
[0051] The term “transfection” refers to the uptake of foreign or exogenous DNA by a cell. A number of transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology, 1973, 52:456; Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2001, supra; Davis et al., Basic Methods in Molecular Biology, 1986, Elsevier; Chu c/ a/., 1981, Gene, 13: 197.
[0052] The term “transduction” refers to the process whereby foreign DNA is introduced into a cell via viral vector. See, e.g., Jones et al., Genetics: Principles and Analysis, 1998, Boston: Jones & Bartlett Publ.
[0053] The terms “polypeptide” or “protein” refer to a macromolecule having the amino acid sequence of a protein, including deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence. The terms “polypeptide” and “protein” specifically encompass antigen-binding molecules, antibodies, or sequences that have deletions from, additions to, and/or substitutions of one or more amino acid of antigenbinding protein. The term “polypeptide fragment” refers to a polypeptide that has an aminoterminal deletion, a carboxyl-terminal deletion, and/or an internal deletion as compared with the full-length native protein. Such fragments can also contain modified amino acids as compared with the native protein. Useful polypeptide fragments include immunologically functional fragments of antigen-binding molecules.
[0054] A “variant” of a polypeptide (e.g., an antigen-binding molecule) comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to another polypeptide sequence. Variants include fusion proteins.
[0055] The term “identity” refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent identity” means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (i.e., an “algorithm”).
[0056] To calculate percent identity, the sequences being compared are typically aligned in a way that gives the largest match between the sequences. One example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., Nucl. Acid Res., 1984, 12, 387; Genetics Computer Group,
University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned for optimal matching of their respective amino acid or nucleotide (the “matched span”, as determined by the algorithm). In certain embodiments, a standard comparison matrix (see, e.g., Dayhoff et al., 1978, Atlas of Protein Sequence and Structure, 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A., 89, 10915-10919 for the BLO-SUM 62 comparison matrix) is also used by the algorithm.
[0057] As used herein, the twenty conventional (e.g., naturally occurring) amino acids and their abbreviations follow conventional usage. See, e.g., Immunology A Synthesis (2nd Edition, Golub and Green, Eds., Sinauer Assoc., Sunderland, Mass. (1991)), which is incorporated herein by reference for any purpose. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as alpha-, alpha-di substituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids can also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, gamma-carboxy-glutamate, epsilon- N,N,N-trimethyllysine, e-N-acetyllysine, O-phosphoserine, N-acetylserine, N- formylmethionine, 3-methylhistidine, 5-hydroxylysine, sigma. -N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.
[0058] Conservative amino acid substitutions can encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties. Naturally occurring residues can be divided into classes based on common side chain properties: a) hydrophobic: norleucine, Met, Ala, Vai, Leu, He; b) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; c) acidic: Asp, Glu; d) basic: His, Lys, Arg;
e) residues that influence chain orientation: Gly, Pro; and f) aromatic: Trp, Tyr, Phe.
[0059] For example, non-conservative substitutions can involve the exchange of a member of one of these classes for a member from another class.
[0060] In making changes to the antigen-binding molecule, the costimulatory or activating domains of the engineered B cell, according to certain embodiments, the hydropathic index of amino acids can be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (- 3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). See, e.g., Kyte et al., 1982, J. Mol. Biol., 157, 105-131. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological embodiments, as in the present case. Exemplary amino acid substitutions are set forth in Table 1.
[0061] The term “derivative” refers to a molecule that includes a chemical modification other than an insertion, deletion, or substitution of amino acids (or nucleic acids). In certain embodiments, derivatives comprise covalent modifications, including, but not limited to, chemical bonding with polymers, lipids, or other organic or inorganic moieties. In certain embodiments, a chemically modified antigen-binding molecule can have a greater circulating half-life than an antigen-binding molecule that is not chemically modified. In some embodiments, a derivative antigen-binding molecule is covalently modified to include one or more water-soluble polymer attachments, including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.
[0062] Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed “peptide mimetics” or “peptidomimetics.” Fauchere, J. L., 1986, Adv. Drug Res., 1986, 15, 29; Veber, D. F. & Freidinger, R. M., 1985, Trends in Neuroscience, 8, 392-396; and Evans, B. E., et al., 1987, J. Med. Chem., 30, 1229-1239, which are incorporated herein by reference for any purpose.
[0063] The term “therapeutically effective amount” refers to a quantity or amount of an agent (e.g., therapeutic cell compositions, immune cells or other therapeutic agent) sufficient to achieve a desired therapeutic effect or response in a subject. Such therapeutically effective amounts are readily ascertained by one of ordinary skill in the art.
[0064] The terms “patient” and “subject” are used interchangeably and include human and non-human animal subjects as well as those with formally diagnosed disorders, those without formally recognized disorders, those receiving medical attention, those at risk of developing the disorders, etc.
[0065] The term “treat” and “treatment” includes therapeutic treatments, prophylactic treatments, and applications in which one reduces the risk that a subject will develop a disorder or other risk factor. Treatment does not require the complete curing of a disorder and encompasses embodiments in which one reduces symptoms or underlying risk factors. The term “prevent” does not require the 100% elimination of the possibility of an event. Rather, it denotes that the likelihood of the occurrence of the event has been reduced in the presence of the compound or method.
[0066] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer’s specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures can be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.
[0067] As used herein, the term “substantially” or “essentially” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% higher compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, the terms “essentially the same” or “substantially the same” refer to a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is about the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
[0068] As used herein, the terms “substantially free of’ and “essentially free of’ are used interchangeably, and when used to describe a composition, such as a cell population or culture media, refer to a composition that is free of a specified substance, such as, 95% free, 96% free, 97% free, 98% free, 99% free of the specified substance, or is undetectable as measured by conventional means. Similar meaning can be applied to the term “absence of,” where referring to the absence of a particular substance or component of a composition.
[0069] As used herein, the term “appreciable” refers to a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length or an event that is readily detectable by one or more standard methods. The terms “not-appreciable” and “not appreciable” and equivalents refer to a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length or an event that is not readily detectable or undetectable by standard methods. In one embodiment, an event is not appreciable if it occurs less than 5%, 4%, 3%, 2%, 1%, 0.1%, 0.001%, or less of the time.
[0070] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises” and “comprising” will be understood to imply the inclusion of stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. In particular embodiments, the terms “include,” “has,” “contains,” and “comprise” are used synonymously.
[0071] As used herein, “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of’. Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present.
[0072] By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that no other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.
[0073] Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[0074] As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In particular embodiments, the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 15%, 10%, 5% or 1%, or any intervening ranges thereof.
[0075] As used herein, the term “introducing” refers to a process that comprises contacting a cell with a polynucleotide, polypeptide, or small molecule. An introducing step may also comprise microinjection of polynucleotides or polypeptides into the cell, use of liposomes to deliver polynucleotides or polypeptides into the cell, or fusion of polynucleotides or polypeptides to cell permeable moi eties to introduce them into a cell.
IL Therapeutic Proteins for Treatment of Fabry Disease
[0076] In various embodiments, the engineered B cell comprises a therapeutic protein to be delivered to a patient in need thereof. As used herein, the term “therapeutic protein” means any protein that may contribute to the treatment, reduction of symptoms, prevention or cure of a disease or disorder in a patient. In certain embodiments, the therapeutic protein may be suitable for treatment of a rare disease or an orphan disease, where said therapy can be achieved by the replacement of a particular protein and/or enzyme. A therapeutic protein may include but is not limited to an enzyme, a ligand, a naturally occurring, engineered and/or chimeric receptor, a cytokine or a chemokine.
[0077] In various embodiments said therapeutic protein is a protein for the treatment of Fabry disease. In various embodiments, the therapeutic protein is a-galactosidase (a-GAL). In various embodiments, the therapeutic protein is human a-GAL. In various embodiments, the therapeutic protein comprises the amino acid sequence of SEQ ID NO. 1. In various embodiments, the therapeutic protein is at least 75%, 80%, 85%, 90% or 95% identical to the amino acid sequence of SEQ ID NO. 1. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence comprising SEQ ID NO. 5. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence comprising a sequence that is at least 75%, 80%, 85%, 90% or 95% identical to the nucleic acid sequence of SEQ ID NO. 5.
[0078] In various embodiments the therapeutic protein comprises the human a-GAL protein, wherein the two leucine residues at the carboxy terminus have been deleted. In various embodiments, the therapeutic protein comprises the amino acid sequence of SEQ ID NO. 2. In various embodiments, the therapeutic protein is at least 75%, 80%, 85%, 90% or 95% identical to the amino acid sequence of SEQ ID NO. 2. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence comprising SEQ ID NO. 6. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence comprising a sequence that is at least 75%, 80%, 85%, 90% or 95% identical to the nucleic acid sequence of SEQ ID NO. 6.
[0079] In various embodiments, the therapeutic protein is a human a-GAL protein, wherein the two leucine residues at the carboxy terminus have been deleted, and the natural human GLA signal peptide has been replaced with an immunoglobulin (Ig) signal peptide. Modification to include an Ig signal peptide directs the a-GAL enzyme towards the secretory pathway, thereby promoting secretion of the expressed therapeutic protein.
[0080] In various embodiments the heavy chain Ig signal peptide is used. In various embodiments, the therapeutic protein comprises the amino acid sequence of SEQ ID NO. 3. In various embodiments, the therapeutic protein is at least 75%, 80%, 85%, 90% or 95% identical to the amino acid sequence of SEQ ID NO. 3. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence comprising SEQ ID NO. 7. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence comprising a sequence that is at least 75%, 80%, 85%, 90% or 95% identical to the nucleic acid sequence of SEQ ID NO. 7.
[0081] In various embodiments the kappa light chain Ig signal peptide is used. In various embodiments, the therapeutic protein comprises the amino acid sequence of SEQ ID NO. 4. In various embodiments, the therapeutic protein is at least 75%, 80%, 85%, 90% or 95% identical to the amino acid sequence of SEQ ID NO. 4. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence comprising SEQ ID NO. 8. In various embodiments, the therapeutic protein is encoded by a nucleic acid sequence comprising a sequence that is at least 75%, 80%, 85%, 90% or 95% identical to the nucleic acid sequence of SEQ ID NO. 8.
III. Cell Therapy for the Treatment of Fabry Disease
[0082] In various embodiments, the therapeutic protein is expressed by engineering a population of B cells to express said therapeutic protein. As used herein, the term “B cell” refers to an immune cell (e.g., a white blood cell or leukocyte) that expresses a B cell receptor (BCR) or produces antibodies. As used herein, the term “B cell” includes any B cell lineage cell type that is derived from a B cell including memory B cells, plasma cells (PCs) and plasmablasts (PBs). A plasmablast is an intermediate, transitional stage cell type that arises during differentiation of activated B cells. It is the immediate precursor to a plasma cell. Both plasmablasts and plasma cells are antibody secreting cells, but mature plasma cells produce and secrete antibodies at a higher level. Plasma cells are the terminal, fully differentiated form resulting from B cell differentiation. The primary function of plasma cells is the robust synthesis and secretion of antibodies. Plasma cells have a highly developed secretory system to support a high level of protein synthesis and secretion. Although some plasma cells are short-lived, others can persist much longer. Antigen-induced B cell activation and differentiation can also result in memory B cells, which can persist for years. Upon a secondary encounter with the same antigen, memory B cells can also proliferate and differentiate into plasma cells. By delivering therapeutic proteins through engineered B cells, the embodiments disclosed herein provide long-term stable expression of the enzyme in vivo. The embodiments disclosed herein, leverage the B cell’s intrinsic protein secretion machinery and its long lifetime in vivo to express therapeutic proteins for treatment of multiple diseases including Fabry disease.
[0083] In various embodiments, the disclosure relates to a population of cells comprising engineered human B cells, wherein human B cells express a therapeutic protein, whose gene has been inserted into either the human AAVS1 or the human ROSA26 locus, wherein the therapeutic protein comprises the human GLA protein.
[0084] In various embodiments said population of cells are for treatment of a patient suffering from Fabry disease. In various embodiments, the population of cells express a therapeutic protein that is a-galactosidase (a-GAL). In various embodiments, the population of cells express a therapeutic protein that is wild type human a-GAL. In various embodiments, the population of cells express a therapeutic protein that comprises the amino acid sequence of SEQ ID NO. 1. In various embodiments, the population of cells express a therapeutic protein that is at least 75%, 80%, 85%, 90% or 95% identical to the amino acid sequence of SEQ ID NO. 1. In various embodiments, the population of cells express a
therapeutic protein that is encoded by a nucleic acid sequence comprising SEQ ID NO. 5. In various embodiments, the population of cells express a therapeutic protein that is encoded by a nucleic acid sequence comprising a sequence that is at least 75%, 80%, 85%, 90% or 95% identical to the nucleic acid sequence of SEQ ID NO. 5.
[0085] In various embodiments, the population of cells express a therapeutic protein comprising the human a-GAL protein, wherein the two leucine residues at the carboxy terminus have been deleted. In various embodiments, the population of cells express a therapeutic protein that comprises the amino acid sequence of SEQ ID NO. 2. In various embodiments, the population of cells express a therapeutic protein that is at least 75%, 80%, 85%, 90% or 95% identical to the amino acid sequence of SEQ ID NO. 2. In various embodiments, the population of cells express a therapeutic protein that is encoded by a nucleic acid sequence comprising SEQ ID NO. 6. In various embodiments, the population of cells express a therapeutic protein that is encoded by a nucleic acid sequence comprising a sequence that is at least 75%, 80%, 85%, 90% or 95% identical to the nucleic acid sequence of SEQ ID NO. 6.
[0086] In various embodiments, the population of cells express a therapeutic protein comprising the human a-GAL protein, wherein the two leucine residues at the carboxy terminus have been deleted, and the natural human a-GAL signal peptide has been replaced with an immunoglobulin (Ig) signal peptide. Modification to include an Ig signal peptide directs the a-GAL enzyme towards the secretory pathway, thereby promoting secretion of the expressed therapeutic protein.
[0087] In various embodiments the heavy chain Ig signal peptide is used. In various embodiments, the population of cells express a therapeutic protein that comprises the amino acid sequence of SEQ ID NO. 3. In various embodiments, the population of cells express a therapeutic protein that is at least 75%, 80%, 85%, 90% or 95% identical to the amino acid sequence of SEQ ID NO. 3. In various embodiments, the population of cells express a therapeutic protein that is encoded by a nucleic acid sequence comprising SEQ ID NO. 7. In various embodiments, the population of cells express a therapeutic protein that is encoded by a nucleic acid sequence comprising a sequence that is at least 75%, 80%, 85%, 90% or 95% identical to the nucleic acid sequence of SEQ ID NO. 7.
[0088] In various embodiments the kappa light chain Ig signal peptide is used. In various embodiments, the population of cells express a therapeutic protein that comprises the amino acid sequence of SEQ ID NO. 4. In various embodiments, the population of cells express a therapeutic protein that is at least 75%, 80%, 85%, 90% or 95% identical to the amino acid sequence of SEQ ID NO. 4. In various embodiments, the population of cells express a therapeutic protein that is encoded by a nucleic acid sequence comprising SEQ ID NO. 8. In various embodiments, the population of cells express a therapeutic protein that is encoded by a nucleic acid sequence comprising a sequence that is at least 75%, 80%, 85%, 90% or 95% identical to the nucleic acid sequence of SEQ ID NO. 8.
[0089] In various embodiments, depending upon the editing protocols used, a certain percentage of the population of cells will express said therapeutic protein. In various embodiments at least 20% of the population of cells express said therapeutic protein. In various embodiments at least 10%, 15%, 20%, 25%, 30%, 35% or 40% of the population of cells express said therapeutic protein.
IV. Methods of Engineering B Cells
[0090] Gene editing (including genomic editing) is a type of genetic engineering in which nucleotide(s)/nucleic acid(s) is/are inserted, deleted, and/or substituted in a DNA sequence, such as in the genome of a targeted cell. Targeted gene editing enables insertion, deletion and/or substitution at pre-selected sites in the genome of a targeted cell (e.g., in a targeted gene or targeted DNA sequence). When a sequence of an endogenous gene is edited, for example by deletion, insertion or substitution of nucleotide(s)/nucleic acid(s), the endogenous gene comprising the affected sequence may be knocked-out or knocked-down due to the sequence alteration. Therefore, targeted editing may be used to disrupt endogenous gene expression. “Targeted integration” refers to a process involving insertion of one or more exogenous sequences, with or without deletion of an endogenous sequence at the insertion site. Targeted integration can result from targeted gene editing when a donor template containing an exogenous sequence is present. As used herein, a “disrupted gene” refers to a gene comprising an insertion, deletion, or substitution relative to an endogenous gene such that expression of a functional protein from the endogenous gene is reduced or inhibited. As used herein, “disrupting a gene” refers to a method of inserting, deleting or substituting at least one nucleotide/nucleic acid in an endogenous gene such that expression of a functional
protein from the endogenous gene is reduced or inhibited. Methods of disrupting a gene are known to those of skill in the art and described herein.
[0091] Targeted editing can be achieved either through a nuclease-independent approach, or through a nuclease - dependent approach. In the nuclease-independent targeted editing approach, homologous recombination is guided by homologous sequences flanking an exogenous polynucleotide to be introduced into an endogenous sequence through the enzymatic machinery of the host cell. The exogenous polynucleotide may introduce deletions, insertions or replacement of nucleotides in the endogenous sequence.
[0092] Alternatively, the nuclease - dependent approach can achieve targeted editing with higher frequency through the specific introduction of double strand breaks (DSBs) by specific rare - cutting nucleases (e.g., endonucleases). Such nuclease - dependent targeted editing also utilizes DNA repair mechanisms, for example, non - homologous end joining (NHEJ), which occurs in response to DSBs. DNA repair by NHEJ often leads to random insertions or deletions (indels) of a small number of endogenous nucleotides. In contrast to NHEJ mediated repair, repair can also occur by a homology directed repair (HDR). When a donor template containing exogenous genetic material flanked by a pair of homology arms is present, the exogenous genetic material can be introduced into the genome by HDR, which results in targeted integration of the exogenous genetic material.
[0093] Available endonucleases capable of introducing specific and targeted DSBs include, but are not limited to, zinc-finger nucleases (ZFN), meganucleases, transcription activator-like effector nucleases (TALEN), and RNA-guided CRISPR Cas9 nuclease (CRISPR/Cas9; Clustered Regular Interspaced Short Palindromic Repeats Associated 9). Additionally, DICE (dual integrase cassette exchange) system utilizing phiC31 and Bxbl integrases may also be used for targeted integration.
[0094] ZFNs are targeted nucleases comprising a nuclease fused to a zinc finger DNA binding domain (ZFBD), which is a polypeptide domain that binds DNA in a sequence specific manner. A zinc finger is a domain of about 30 amino acids within the zinc fingerbinding domain whose structure is stabilized through coordination of a zinc ion. Examples of zinc fingers include, but not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. A designed zinc finger domain is a domain not occurring in nature whose design/composition results principally from rational criteria, e.g., application of substitution
rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496. A selected zinc finger domain is a domain not found in nature whose production results primarily from an empirical process such as phage display, interaction trap or hybrid selection. ZFNs are described in greater detail in U.S. Pat. Nos. 7,888,121 and 7,972,854. The most recognized example of a ZFN is a fusion of the Fokl nuclease with a zinc finger DNA binding domain.
[0095] A TALEN is a targeted nuclease comprising a nuclease fused to a TAL effector DNA binding domain. A “transcription activator-like effector DNA binding domain”, “TAL effector DNA binding domain", or “TALE DNA binding domain” is a polypeptide domain of TAL effector proteins that is responsible for binding of the TAL effector protein to DNA. TAL effector proteins are secreted by plant pathogens of the genus Xanthomonas during infection. These proteins enter the nucleus of the plant cell, bind effector-specific DNA sequences via their DNA binding domain, and activate gene transcription at these sequences via their transactivation domains. TAL effector DNA binding domain specificity depends on an effector - variable number of imperfect 34 amino acid repeats, which comprise polymorphisms at select repeat positions called repeat variable diresidues (RVD). TALENs are described in greater detail in US Patent Application No. 2011/0145940. The most recognized example of a TALEN in the art is a fusion polypeptide of the Fokl nuclease to a TAL effector DNA binding domain.
[0096] Additional examples of targeted nucleases suitable for use as provided herein include, but are not limited to, Bxbl, phiC31, R4, PhiBTl, and WB/SPBc/TP901-l, whether used individually or in combination.
[0097] Other non - limiting examples of targeted nucleases include naturally - occurring and recombinant nucleases, e.g., CRISPR/Cas9, restriction endonucleases, meganucleases homing endonucleases, and the like.
1. CRISPR-Cas9 Gene Editing
[0098] The CRISPR-Cas9 system is a naturally-occurring defense mechanism in prokaryotes that has been repurposed as an RNA-guided DNA - targeting platform used for gene editing. It relies on the DNA nuclease Cas9, and two noncoding RNAs, crisprRNA
(CrRNA) and trans-activating RNA (tracrRNA), to target the cleavage of DNA. CRISPR is an abbreviation for Clustered Regularly Interspaced Short Palindromic Repeats, a family of DNA sequences found in the genomes of bacteria and archaea that contain fragments of DNA (spacer DNA) with similarity to foreign DNA previously exposed to the cell, for example, by viruses that have infected or attacked the prokaryote. These fragments of DNA are used by the prokaryote to detect and destroy similar foreign DNA upon reintroduction, for example, from similar viruses during subsequent attacks. Transcription of the CRISPR locus results in the formation of an RNA molecule comprising the spacer sequence, which associates with and targets Cas (CRISPR-associated) proteins able to recognize and cut the foreign, exogenous DNA. Numerous types and classes of CRISPR/Cas systems have been described (see, e.g., Koonin et al., (2017) Curr Opin Microbiol 37:67-78).
[0099] crRNA drives sequence recognition and specificity of the CRISPR - Cas9 complex through Watson - Crick base pairing typically with a 20 nucleotide (nt) sequence in the target DNA. Changing the sequence of the 5 ' 20nt in the crRNA allows targeting of the CRISPR - Cas9 complex to specific loci. The CRISPR - Cas9 complex only binds DNA sequences that contain a sequence match to the first 20 nt of the crRNA, single - guide RNA (sgRNA), if the target sequence is followed by a specific short DNA motif (with the sequence NGG) referred to as a protospacer adjacent motif (PAM).
[0100] TracrRNA hybridizes with the 3' end of crRNA to form an RNA-duplex structure that is bound by the Cas9 endonuclease to form the catalytically active CRISPR-Cas9 complex, which can then cleave the target DNA.
[0101] Once the CRISPR - Cas9 complex is bound to DNA at a target site, two independent nuclease domains within the Cas9 enzyme each cleave one of the DNA strands upstream of the PAM site, leaving a double-strand break (DSB) where both strands of the DNA terminate in a base pair (a blunt end).
[0102] After binding of CRISPR - Cas9 complex to DNA at a specific target site and formation of the site - specific DSB, the next key step is repair of the DSB. Cells use two main DNA repair pathways to repair the DSB: non - homologous end - joining (NHEJ) and homology - directed repair (HDR).
[0103] NHEJ is a robust repair mechanism that appears highly active in the majority of cell types, including nondividing cells. NHEJ is error-prone and can often result in the removal or addition of between one and several hundred nucleotides at the site of the DSB, though such modifications are typically < 20 nt. The resulting insertions and deletions (indels) can disrupt coding or noncoding regions of genes. Alternatively, HDR uses a long stretch of homologous donor DNA, provided endogenously or exogenously, to repair the DSB with high fidelity. HDR is active only in dividing cells, and occurs at a relatively low frequency in most cell types. In many embodiments of the present disclosure, NHEJ is utilized as the repair operant.
[0104] In some embodiments, the Cas9 (CRISPR associated protein 9) endonuclease is from Streptococcus pyogenes, although other Cas9 homologs may be used. It should be understood, that wild - type Cas9 may be used or modified versions of Cas9 may be used (e.g., evolved versions of Cas9, or Cas9 orthologues or variants), as provided herein. In some embodiments, Cas9 may be substituted with another RNA- guided endonuclease, such as Cpfl (of a class II CRISPR/Cas system).
[0105] In some embodiments, the CRISPR/Cas system comprise components derived from a Type-1, Type-II, or Type-III system. Updated classification schemes for CRISPR/Cas loci define Class 1 and Class 2 CRISPR/Cas systems, having Types Ito V or VI (Makarova et al., (2015) Nat Rev Microbiol, 13(11):722-36; Shmakov etal., (2015)) Mol Cell, 60:385- 397). Class 2 CRISPR / Cas systems have single protein effectors. Cas proteins of Types II, V, and VI are single - protein, RNA - guided endonucleases, herein called “Class 2 Cas nucleases.” Class 2 Cas nucleases include, for example, Cas9, Cpfl, C2cl, C2c2, and C2c3 proteins. The Cpfl nuclease (Zetsche et al., (2015) Cell 163: 1-13) is homologous to Cas9, and contains a RuvC - like nuclease domain.
[0106] In some embodiments, the Cas nuclease is from a Type - II CRISPR/Cas system (e.g., a Cas9 protein from a CRISPR / Cas9 system). In some embodiments, the Cas nuclease is from a Class 2 CRISPR Cas system (a single protein Cas nuclease such as a Cas9 protein or a Cpfl protein). The Cas9 and Cpfl family of proteins are enzymes with DNA endonuclease activity, and they can be directed to cleave a desired nucleic acid target by designing an appropriate guide RNA, as described further herein.
[0107] In some embodiments, a Cas nuclease may comprise more than one nuclease domain. For example, a Cas9 nuclease may comprise at least one RuvC-like nuclease domain (e.g., Cpfl) and at least one HNH-like nuclease domain (e.g., Cas9). In some embodiments, the Cas9 nuclease introduces a DSB in the target sequence. In some embodiments, the Cas9 nuclease is modified to contain only one functional nuclease domain. For example, the Cas9 nuclease is modified such that one of the nuclease domains is mutated or fully or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, the Cas9 nuclease is modified to contain no functional RuvC-like nuclease domain. In other embodiments, the Cas9 nuclease is modified to contain no functional HNH- like nuclease domain. In some embodiments in which only one of the nuclease domains is functional, the Cas9 nuclease is a nickase that is capable of introducing a single-stranded break (a “nick”) into the target sequence. In some embodiments, a conserved amino acid within a Cas9 nuclease domain is substituted to reduce or alter a nuclease activity. In some embodiments, the Cas nuclease nickase comprises an amino acid substitution in the RuvC- like nuclease domain. Exemplary amino acid substitutions in the RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 nuclease). In some embodiments, the nickase comprises an amino acid substitution in the HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH-like nuclease domain include E762A, H840A, N863 A, H983 A, and D986A (based on the S. pyogenes Cas9 nuclease).
[0108] In some embodiments, the Cas nuclease is from a Type-I CRISPR/Cas system. In some embodiments, the Cas nuclease is a component of the Cascade complex of a Type-I CRISPR/Cas system. For example, the Cas nuclease is a Cas3 nuclease. In some embodiments, the Cas nuclease is derived from a Type-III CRISPR/Cas system. In some embodiments, the Cas nuclease is derived from Type-IV CRISPR/Cas system. In some embodiments, the Cas nuclease is derived from a Type-V CRISPR/Cas system. In some embodiments, the Cas nuclease is derived from a Type- VI CRISPR/Cas system.
2. Guide RNAs
[0109] The present disclosure provides a genome-targeting nucleic acid that can direct the activities of an associated polypeptide (e.g., a site-directed polypeptide) to a specific target sequence within a target nucleic acid. The genome-targeting nucleic acid can be an RNA. A genome-targeting RNA is referred to as a “guide RNA” or “gRNA” herein. A guide RNA comprises at least a spacer sequence that hybridizes to a target nucleic acid
sequence of interest, and a CRISPR repeat sequence. In Type II systems, the gRNA also comprises a second RNA called the tracrRNA sequence. In the Type II gRNA, the CRISPR repeat sequence and tracrRNA sequence hybridize to each other to form a duplex. In the Type V gRNA, the crRNA forms a duplex. In both systems, the duplex binds a site-directed polypeptide, such that the guide RNA and site-direct polypeptide form a complex. In some embodiments, the genome-targeting nucleic acid provides target specificity to the complex by virtue of its association with the site-directed polypeptide. The genome-targeting nucleic acid thus directs the activity of the site-directed polypeptide.
[0110] As is understood by the person of ordinary skill in the art, each guide RNA is designed to include a spacer sequence complementary to its genomic target sequence. See Jinek et a/., Science, 337, 816-821 (2012) and Deltcheva et al., Nature, 471, 602-607 (2011).
[OHl] In some embodiments, the genome-targeting nucleic acid (e.g., gRNA) is a double-molecule guide RNA. In some embodiments, the genome-targeting nucleic acid (e.g., gRNA) is a single-molecule guide RNA.
[0112] A double-molecule guide RNA comprises two strands of RNA. The first strand comprises in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence and a minimum CRISPR repeat sequence. The second strand comprises a minimum tracrRNA sequence (complementary to the minimum CRISPR repeat sequence), a 3' tracrRNA sequence and an optional tracrRNA extension sequence.
[0113] A single-molecule guide RNA (referred to as a “sgRNA”) in a Type II system comprises, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single-molecule guide linker, a minimum tracrRNA sequence, a 3' tracrRNA sequence and an optional tracrRNA extension sequence. The optional tracrRNA extension may comprise elements that contribute additional functionality (e.g., stability) to the guide RNA. The single-molecule guide linker links the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension comprises one or more hairpins.
[0114] A single-molecule guide RNA in a Type V system comprises, in the 5' to 3' direction, a minimum CRISPR repeat sequence and a spacer sequence.
[0115] In some embodiments, the sgRNA comprises a 20 nucleotide spacer sequence at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA comprises a less than 20 nucleotide spacer sequence at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA comprises a more than 20 nucleotide spacer sequence at the 5' end of the sgRNA sequence.
[0116] In some embodiments, the sgRNA comprises comprise no uracil at the 3' end of the sgRNA sequence. In some embodiments, the sgRNA comprises comprise one or more uracil at the 3' end of the sgRNA sequence. For example, the sgRNA can comprise 1 uracil (U) at the 3' end of the sgRNA sequence. The sgRNA can comprise 2 uracil (UU) at the 3' end of the sgRNA sequence. The sgRNA can comprise 3 uracil (UUU) at the 3' end of the sgRNA sequence. The sgRNA can comprise 4 uracil (UUUU) at the 3' end of the sgRNA sequence. The sgRNA can comprise 5 uracil (UUUUU) at the 3' end of the sgRNA sequence. The sgRNA can comprise 6 uracil (UUUUUU) at the 3' end of the sgRNA sequence. The sgRNA can comprise 7 uracil (UUUUUUU) at the 3' end of the sgRNA sequence. The sgRNA can comprise 8 uracil (UUUUUUUU) at the 3' end of the sgRNA sequence.
[0117] The sgRNA can be unmodified or modified. For example, modified sgRNAs can comprise one or more 2'-O-methyl phosphorothioate nucleotides.
[0118] By way of illustration, guide RNAs used in the CRISPR/Cas/Cpfl system, or other smaller RNAs can be readily synthesized by chemical means, as illustrated below and described in the art. While chemical synthetic procedures are continually expanding, purifications of such RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more challenging as polynucleotide lengths increase significantly beyond a hundred or so nucleotides. One approach used for generating RNAs of greater length is to produce two or more molecules that are ligated together. Much longer RNAs, such as those encoding a Cas9 or Cpfl endonuclease, are more readily generated enzymatically. Various types of RNA modifications can be introduced during or after chemical synthesis and/or enzymatic generation of RNAs, e.g., modifications that enhance stability, reduce the likelihood or degree of innate immune response, and/or enhance other attributes, as described in the art.
[0119] In some embodiments, indel frequency (editing frequency) may be determined using a TIDE analysis, which can be used to identify highly efficient gRNA molecules. In
some embodiments, a highly efficient gRNA yields a gene editing frequency of higher than 80%. For example, a gRNA is considered to be highly efficient if it yields a gene editing frequency of at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
[0120] In some embodiments, gene disruption may occur by deletion of a genomic sequence using two guide RNAs. Methods of using CRISPR-Cas gene editing technology to create a genomic deletion in a cell (e.g., to knock out a gene in a cell) are known (Bauer D E et al. Vis. Exp. 2015; 95;e52118).
3. Spacer Sequence
[0121] In some embodiments, a gRNA comprises a spacer sequence. A spacer sequence is a sequence (e.g., a 20 nucleotide sequence) that defines the target sequence (e.g., a DNA target sequences, such as a genomic target sequence) of a target nucleic acid of interest. In some embodiments, the spacer sequence is 15 to 30 nucleotides. In some embodiments, the spacer sequence is 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, a spacer sequence is 20 nucleotides.
[0122] The “target sequence” is adjacent to a PAM sequence and is the sequence modified by an RNA-guided nuclease (e.g., Cas9). The “target nucleic acid” is a doublestranded molecule: one strand comprises the target sequence and is referred to as the “PAM strand,” and the other complementary strand is referred to as the “non-PAM strand.” One of skill in the art recognizes that the gRNA spacer sequence hybridizes to the reverse complement of the target sequence, which is located in the non-PAM strand of the target nucleic acid of interest. Thus, the gRNA spacer sequence is the RNA equivalent of the target sequence. For example, if the target sequence is 5'-AGAGCAACAGTGCTGTGGCC-3', then the gRNA spacer sequence is 5'-AGAGCAACAGUGCUGUGGCC-3'. The spacer of a gRNA interacts with a target nucleic acid of interest in a sequence-specific manner via hybridization (z.e., base pairing). The nucleotide sequence of the spacer thus varies depending on the target sequence of the target nucleic acid of interest.
[0123] In a CRISPR/Cas system herein, the spacer sequence is designed to hybridize to a region of the target nucleic acid that is located 5' of a PAM of the Cas9 enzyme used in the system. The spacer may perfectly match the target sequence or may have mismatches. Each Cas9 enzyme has a particular PAM sequence that it recognizes in a target DNA. For example, S. pyogenes recognizes in a target nucleic acid a PAM that comprises the sequence
5'-NRG-3', where R comprises either A or G, where N is any nucleotide and N is immediately 3' of the target nucleic acid sequence targeted by the spacer sequence.
[0124] In some embodiments, the target nucleic acid sequence comprises 20 nucleotides. In some embodiments, the target nucleic acid comprises less than 20 nucleotides. In some embodiments, the target nucleic acid comprises more than 20 nucleotides. In some embodiments, the target nucleic acid comprises at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. In some embodiments, the target nucleic acid comprises at most: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. In some embodiments, the target nucleic acid sequence comprises 20 bases immediately 5' of the first nucleotide of the PAM. For example, in a sequence comprising 5'- NNNNNNNNNNNNNNNNNNNNNRG-3', the target nucleic acid comprises the sequence that corresponds to the Ns, wherein N is any nucleotide, and the underlined NRG sequence is the S. pyogenes PAM.
4. Methods of Making gRNAs
[0125] The gRNAs of the present disclosure are produced by a suitable means available in the art, including but not limited to in vitro transcription (IVT), synthetic and/or chemical synthesis methods, or a combination thereof. Enzymatic (IVT), solid-phase, liquid-phase, combined synthetic methods, small region synthesis, and ligation methods are utilized. In one embodiment, the gRNAs are made using IVT enzymatic synthesis methods. Methods of making polynucleotides by IVT are known in the art and are described in International Application PCT/US2013/30062. Accordingly, the present disclosure also includes polynucleotides, e.g., DNA, constructs and vectors are used to in vitro transcribe a gRNA described herein.
[0126] In some embodiments, non-natural modified nucleobases are introduced into polynucleotides, e.g., gRNA, during synthesis or post-synthesis. In certain embodiments, modifications are on intemucleoside linkages, purine or pyrimidine bases, or sugar. In some embodiments, a modification is introduced at the terminal of a polynucleotide; with chemical synthesis or with a polymerase enzyme. Examples of modified nucleic acids and their synthesis are disclosed in PCT application No. PCT/US2012/058519. Synthesis of modified polynucleotides is also described in Verma and Eckstein, Annual Review of Biochemistry, vol. 76, 99-134 (1998).
[0127] In some embodiments, enzymatic or chemical ligation methods are used to conjugate polynucleotides or their regions with different functional moieties, such as targeting or delivery agents, fluorescent labels, liquids, nanoparticles, etc. Conjugates of polynucleotides and modified polynucleotides are reviewed in Goodchild, Bioconjugate Chemistry, vol. 1(3), 165-187 (1990).
[0128] Certain embodiments of the invention also provide nucleic acids, e.g., vectors, encoding gRNAs described herein. In some embodiments, the nucleic acid is a DNA molecule. In other embodiments, the nucleic acid is an RNA molecule. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a crRNA. In some embodiments, the nucleotide sequence encoding the crRNA comprises a spacer flanked by all or a portion of a repeat sequence from a naturally-occurring CRISPR/Cas system. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a tracrRNA. In some embodiments, the crRNA and the tracrRNA is encoded by two separate nucleic acids. In other embodiments, the crRNA and the tracrRNA is encoded by a single nucleic acid. In some embodiments, the crRNA and the tracrRNA is encoded by opposite strands of a single nucleic acid. In other embodiments, the crRNA and the tracrRNA is encoded by the same strand of a single nucleic acid.
[0129] In some embodiments, the gRNAs provided by the disclosure are chemically synthesized by any means described in the art (see e.g., WO/2005/01248). While chemical synthetic procedures are continually expanding, purifications of such RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more challenging as polynucleotide lengths increase significantly beyond a hundred or so nucleotides. One approach used for generating RNAs of greater length is to produce two or more molecules that are ligated together.
[0130] In some embodiments, the gRNAs provided by the disclosure are synthesized by enzymatic methods (e.g., in vitro transcription, IVT).
[0131] Various types of RNA modifications can be introduced during or after chemical synthesis and/or enzymatic generation of RNAs, e.g., modifications that enhance stability, reduce the likelihood or degree of innate immune response, and/or enhance other attributes, as described in the art.
[0132] In certain embodiments, more than one guide RNA can be used with a CRISPR/Cas nuclease system. Each guide RNA may contain a different targeting sequence, such that the CRISPR/Cas system cleaves more than one target nucleic acid. In some embodiments, one or more guide RNAs may have the same or differing properties such as activity or stability within the Cas9 RNP complex. Where more than one guide RNA is used, each guide RNA can be encoded on the same or on different vectors. The promoters used to drive expression of the more than one guide RNA is the same or different.
[0133] The guide RNA may target any sequence of interest via the targeting sequence (e.g., spacer sequence) of the crRNA. In some embodiments, the degree of complementarity between the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule is about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule is 100% complementary. In other embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain at least one mismatch. For example, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain 1-6 mismatches. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain 5 or 6 mismatches.
[0134] The length of the targeting sequence may depend on the CRISPR/Cas9 system and components used. For example, different Cas9 proteins from different bacterial species have varying optimal targeting sequence lengths. Accordingly, the targeting sequence may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length. In some embodiments, the targeting sequence may comprise 18-24 nucleotides in length. In some embodiments, the targeting sequence may comprise 19-21 nucleotides in length. In some embodiments, the targeting sequence may comprise 20 nucleotides in length.
[0135] In some embodiments of the present disclosure, a CRISPR/Cas nuclease system includes at least one guide RNA. In some embodiments, the guide RNA and the Cas protein may form a ribonucleoprotein (RNP), e.g., a CRISPR/Cas complex. The guide RNA may
guide the Cas protein to a target sequence on a target nucleic acid molecule (e.g., a genomic DNA molecule), where the Cas protein cleaves the target nucleic acid. In some embodiments, the CRISPR/Cas complex is a Cpfl /guide RNA complex. In some embodiments, the CRISPR complex is a Type-II CRISPR/Cas9 complex. In some embodiments, the Cas protein is a Cas9 protein. In some embodiments, the CRISPR/Cas9 complex is a Cas9/guide RNA complex.
5. Delivery of guide RNA and Nuclease
[0136] In some embodiments, a gRNA and an RNA-guided nuclease are delivered to a cell separately, either simultaneously or sequentially. In some embodiments, a gRNA and an RNA-guided nuclease are delivered to a cell together. In some embodiments, a gRNA and an RNA-guided nuclease are pre-complexed together to form a ribonucleoprotein (RNP).
[0137] RNPs are useful for gene editing, at least because they minimize the risk of promiscuous interactions in a nucleic acid-rich cellular environment and protect the RNA from degradation. Methods for forming RNPs are known in the art. In some embodiments, an RNP containing an RNA-guided nuclease e.g., a Cas nuclease, such as a Cas9 nuclease) and a gRNA targeting a gene of interest is delivered a cell (e.g.: a T cell). In some embodiments, an RNP is delivered to a T cell by electroporation.
[0138] As used herein, a “AAVS1 or ROSA26 targeting RNP” refers to a gRNA that targets the AAVS1 or ROSA26 genes pre-complexed with an RNA-guided nuclease. In some embodiments, a AAVS1 or ROSA26 targeting RNP is delivered to a cell. In some embodiments, more than one RNP is delivered to a cell. In some embodiments, more than one RNA is delivered to a cell separately. In some embodiments, more than one RNP is delivered to the cell simultaneously.
[0139] In some embodiments, an RNA-guided nuclease is delivered to a cell in a DNA vector that expresses the RNA-guided nuclease, an RNA that encodes the RNA-guided nuclease, or a protein. In some embodiments, a gRNA targeting a gene is delivered to a cell as an RNA, or a DNA vector that expresses the gRNA.
[0140] Delivery of an RNA-guided nuclease, gRNA, and/or an RNP may be through direct injection or cell transfection using known methods, for example, electroporation or chemical transfection. Other cell transfection methods may be used.
6. Multi-Modal or Differential Delivery of Components
[0141] Skilled artisans will appreciate that different components of genome editing systems can be delivered together or separately and simultaneously or nonsimultaneously. Separate and/or asynchronous delivery of genome editing system components may be particularly desirable to provide temporal or spatial control over the function of genome editing systems and to limit certain effects caused by their activity.
[0142] Different or differential modes as used herein refer to modes of delivery that confer different pharmacodynamic or pharmacokinetic properties on the subject component molecule, e.g, a RNA-guided nuclease molecule, gRNA, template nucleic acid, or payload. For example, the modes of delivery can result in different tissue distribution, different halflife, or different temporal distribution, e.g, in a selected compartment, tissue, or organ.
[0143] Some modes of delivery, e.g., delivery by a nucleic acid vector that persists in a cell, or in progeny of a cell, e.g., by autonomous replication or insertion into cellular nucleic acid, result in more persistent expression of and presence of a component. Examples include viral, e.g., AAV or lentivirus, delivery.
[0144] By way of example, the components of a genome editing system, e.g., a RNA- guided nuclease and a gRNA, can be delivered by modes that differ in terms of resulting halflife or persistent of the delivered component the body, or in a particular compartment, tissue or organ. In an embodiment, a gRNA can be delivered by such modes. The RNA-guided nuclease molecule component can be delivered by a mode which results in less persistence or less exposure to the body or a particular compartment or tissue or organ.
[0145] More generally, in an embodiment, a first mode of delivery is used to deliver a first component and a second mode of delivery is used to deliver a second component. The first mode of delivery confers a first pharmacodynamic or pharmacokinetic property. The first pharmacodynamic property can be, e.g., distribution, persistence, or exposure, of the component, or of a nucleic acid that encodes the component, in the body, a compartment, tissue or organ. The second mode of delivery confers a second pharmacodynamic or pharmacokinetic property. The second pharmacodynamic property can be, e.g., distribution, persistence, or exposure, of the component, or of a nucleic acid that encodes the component, in the body, a compartment, tissue or organ.
[0146] In certain embodiments, the first pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure, is more limited than the second pharmacodynamic or pharmacokinetic property.
[0147] In certain embodiments, the first mode of delivery is selected to optimize, e.g., minimize, a pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure.
[0148] In certain embodiments, the second mode of delivery is selected to optimize, e.g., maximize, a pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure.
[0149] In certain embodiments, the first mode of delivery comprises the use of a relatively persistent element, e.g., a nucleic acid, e.g., a plasmid or viral vector, e.g., an AAV, adenovirus or lentivirus. As such vectors are relatively persistent product transcribed from them would be relatively persistent.
[0150] In certain embodiments, the second mode of delivery comprises a relatively transient element, e.g., an RNA or protein.
[0151] In certain embodiments, the first component comprises gRNA, and the delivery mode is relatively persistent, e.g., the gRNA is transcribed from a plasmid or viral vector, e.g., an AAV, adenovirus or lentivirus. Transcription of these genes would be of little physiological consequence because the genes do not encode for a protein product, and the gRNAs are incapable of acting in isolation. The second component, a RNA-guided nuclease molecule, is delivered in a transient manner, for example as mRNA encoding the protein or as protein, ensuring that the full RNA-guided nuclease molecule/gRNA complex is only present and active for a short period of time.
[0152] Furthermore, the components can be delivered in different molecular form or with different delivery vectors that complement one another to enhance safety and tissue specificity.
[0153] Use of differential delivery modes can enhance performance, safety, and/or efficacy, e.g., the likelihood of an eventual off-target modification can be reduced. Delivery of immunogenic components, e.g., Cas9 molecules, by less persistent modes can reduce
immunogenicity, as peptides from the bacterially-derived Cas enzyme are displayed on the surface of the cell by WIC molecules. A two-part delivery system can alleviate these drawbacks.
[0154] Differential delivery modes can be used to deliver components to different, but overlapping target regions. The formation active complex is minimized outside the overlap of the target regions. Thus, in an embodiment, a first component, e.g., a gRNA is delivered by a first delivery mode that results in a first spatial, e.g., tissue, distribution. A second component, e.g., a RNA-guided nuclease molecule is delivered by a second delivery mode that results in a second spatial, e.g., tissue, distribution. In an embodiment the first mode comprises a first element selected from a liposome, nanoparticle, e.g., polymeric nanoparticle, and a nucleic acid, e.g., viral vector. The second mode comprises a second element selected from the group. In an embodiment, the first mode of delivery comprises a first targeting element, e.g., a cell specific receptor or an antibody, and the second mode of delivery does not include that element. In certain embodiments, the second mode of delivery comprises a second targeting element, e.g., a second cell specific receptor or second antibody.
[0155] When the RNA-guided nuclease molecule is delivered in a virus delivery vector, a liposome, or polymeric nanoparticle, there is the potential for delivery to and therapeutic activity in multiple tissues, when it may be desirable to only target a single tissue. A two-part delivery system can resolve this challenge and enhance tissue specificity. If the gRNA and the RNA-guided nuclease molecule are packaged in separated delivery vehicles with distinct but overlapping tissue tropism, the fully functional complex is only be formed in the tissue that is targeted by both vectors.
V. Methods of Treatment
[0156] In various aspects of the invention, the engineered B cells will be delivered as a therapeutic to a patient in need thereof. In various embodiments, the engineered B cells will be capable of treating or preventing various diseases or disorders relating to Fabry disease.
[0157] In some respects, the invention comprises a pharmaceutical composition comprising a population of gene edited B cells comprising at least one therapeutic protein as described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an additional active agent.
[0158] In one aspect, pharmaceutical compositions according to the invention are administered to a subject in a dosage sufficient to achieve a therapeutic effect. As used herein “therapeutic” effect or action includes an effect or action of a pharmaceutical composition of the invention intended to cure, mitigate, treat, or prevent, or stabilize the progression of, a disease, disorder or condition, or affect the structure or any function of the body of a subject. It will be appreciated that appropriate doses and dosing schedules may be vary according to the subject and the intended therapeutic effect, and may dependent upon such factors as, for example, the disease, disorder or condition being treated, and the general health, age, body weight, sex, or relevant genetic makeup of the subject. Such appropriate dose levels and dosing schedules can be determined by the healthcare provider as needed. Additionally, multiple doses of engineered B cells can be provided in accordance with the invention.
[0159] In some embodiments, the expanded population of engineered B cells are autologous B cells. In some embodiments, the engineered B cells are allogeneic B cells. In some embodiments, the engineered B cells are heterologous B cells. In some embodiments, the engineered B cells of the present application are prepared by in vivo transfection or in vivo transduction. In other embodiments, the engineered cells are prepared ex vivo by transfection or transduction.
[0160] As used herein, the term “subject” or “patient” means an individual. In some aspect, a subject is a mammal such as a human. In some aspect, a subject can be a nonhuman primate. Non-human primates include marmosets, monkeys, chimpanzees, gorillas, orangutans, and gibbons, to name a few. The term “subject” also includes domesticated animals, such as cats, dogs, etc., livestock (e.g., llama, horses, cows), wild animals (e.g., deer, elk, moose, etc.,), laboratory animals (e.g., mouse, rabbit, rat, gerbil, guinea pig, etc.) and avian species (e.g, chickens, turkeys, ducks, etc.). Preferably, the subject is a human subject. More preferably, the subject is a human patient.
[0161] In additional embodiments, the composition comprising gene edited B cells can be administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate. Exemplary NSAIDs include ibuprofen,
naproxen, naproxen sodium, Cox-2 inhibitors, and sialylates. Exemplary analgesics include acetaminophen, oxycodone, tramadol of proporxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as the TNF antagonists, (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®) and infliximab (REMICADE®)), chemokine inhibitors and adhesion molecule inhibitors. The biological response modifiers include monoclonal antibodies as well as recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular) and minocycline.
[0162] In certain embodiments, the compositions described herein are administered in conjunction with a cytokine. “Cytokine” as used herein is meant to refer to proteins released by one cell population that act on another cell as intercellular mediators. Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors (NGFs) such as NGF-beta; platelet-growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, beta, and -gamma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (Ils) such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumor necrosis factor such as TNF-alpha or TNF -beta; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of the native sequence cytokines.
[0163] In some aspects, the method further comprises administering at least a second therapy to the subject. In various embodiments, the method further comprises a second therapy for the treatment of Fabry disease. In certain aspects, wherein the therapy is an oral chaperone therapy. In some embodiments, the second therapy is Galafold® (migalastat). In various embodiments the second therapy is enzyme replacement therapy. In various embodiments the enzyme replacement therapy is an infusion of recombinant a-GAL. In various embodiments, the second therapy is a substrate reduction therapy. In various embodiments, the second therapy is a therapy to reduce how much Gb3 is made by blocking other enzymes necessary for its production.
EXAMPLES
[0164] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.
Example 1. Targeted Editing of B Cells
[0165] Human B cells were isolated from healthy donors using magnetic beads and activated using CD40 ligand and IL21. B cells were engineered using an rAAV6 encoding a- GAL protein and either an AAVS1 specific or ROSA26 specific RNP. See FIG. 1 A (AAVS1) and FIG. IB (ROSA26). B cells were edited to express the WT human a-GAL protein (1137, SEQ ID No. 9), the a-GAL protein with a carboxy -terminal LL truncation of the human a-GAL protein (1138, SEQ ID No. 10), or a a-GAL protein with both the carboxy-terminal LL truncation and the addition of a heavy chain (1139, SEQ ID No. 13) or light chain (1140, SEQ ID No. 11) signal peptide. As a control, B cells were only transduced with rAAV6 in absence of RNP electroporation to determine the level of a-GAL activity from AAV6 episomal expression in B cells (see, e.g., FIG. 2A-B “Episomal”).
[0166] Human B cell isolation, activation, and expansion. Buffy coats from healthy donors were obtained from Stanford Blood Center (Menlo Park, CA, USA). PBMCs were isolated from buffy coats using Ficoll-Paque (GE Healthcare, Chicago, IL). Primary human
B cells were isolated using the EASYSEP™ Human B Cell Isolation Kit according to manufacturer’s instruction (STEMCELL Technologies Inc., Cambridge, MA, USA). B cells were isolated from peripheral blood of healthy donors and cultured for 3 days in activation cocktail (CD40L, CpG, IL2, IL 10, IL 15 and IL21) for 3 days. At day 3, gene-editing was performed using Cas9/sgRNA delivered via electroporation and AAV6 transduction. After gene editing, B cells were cultured in activation media for an additional 6 days.
[0167] AAVS1, ROSA26 sgRNAs and CRISPR engineering. A chemically modified sgRNA oligomer targeting AA VS1 or ROSA26 was manufactured by IDT (Integrated DNA Technologies, Coralville, Iowa, USA). Recombinant S. pyogenes Cas9 enzyme was purchased from IDT (Integrated DNA Technologies, Coralville, Iowa, USA). Cas9 was incubated with sgRNA at a molar ratio of 1 : 1.6 at room temperature for 10 minutes prior to mixing with B cells. Engineering of primary human B cells was carried out using an AMAXA™ 4D-NUCLEOF ACTOR™ in P3 nucleofection solution with program CM-137 (Lonza, Basel, Switzerland). 100 pmol RNP was used for electroporation with 1 million activated human B cells in 20 pl volume. Immediately after electroporation, B cells were transduced with rAAV6 donor at a multiplicity of infection (MOI) of 100,000 viral genomes (vg)/pl to maximize efficiency of transduction.
[0168] Measurement of a-GAL activity. Activity of secreted a-GAL forms from gene edited human B cells differentiated to PBs or PCs was then measured. Supernatants from edited human B cells were collected at 6, 9 and 12 days after gene editing. A-GAL specific activity was measured using the Alpha Galactosidase Activity Assay kit according to manufacturer’s instruction (Abeam, ab239716).
[0169] Results. As is shown in FIGs. 2A and 2B, when the human GLA gene was inserted into either the AAVS1 or ROSA26 loci, the resulting supernatant from these edited B cells showed increased a-GAL activity as compared to controls. This indicates that edited cells demonstrate increased expression and secretion of the human a-GAL protein. B cells edited with hGLA both the carboxy -terminal LL truncation and the addition of a heavy chain (SEQ ID No. 3) or light chain (SEQ ID No. 4) signal peptide showed the greatest increase in a-GAL activity.
Example 2. A-GAL Activity in Differentiated Plasma Blasts and Plasma Cells
[0170] Next, human B cells isolated from peripheral blood were edited at the AAVS1 locus to again express the WT ha-GAL protein (SEQ ID No. 1), the a-GAL protein with a carboxy-terminal LL truncation of the ha-GAL protein (SEQ ID No. 2), or a a-GAL protein with both the carboxy-terminal LL truncation and the addition of a heavy chain (SEQ ID No. 3) or light chain (SEQ ID No. 4) signal peptide. As a control, B cells were only transduced with rAAV6 vectors, without RNP nucleofection (“episomal”). The B cells were next differentiated toward either PBs or PCs, and a-GAL activity was measured using the protocol described in Example 1.
[0171] Differentiation to PBs and PCs. Human B cells activated and expanded B cells were culture in a media containing IL2, IL6, IL10, IL15, IL21 for 3 days. This process leads to a mixture of plasmablasts and B cells. B cells were further differentiated into a mixture of B cells, plasmablasts, and plasma cells via 3 -day incubation in a media containing IL6, IL15, IL21, and INF-a.
[0172] Three days after media change, the supernatant from activated B cells and the differentiated PBs and PCs was measured for a-GAL activity. A-Gal activity was measured using the protocol described in Example 1.
[0173] Results. As is shown in FIG. 3, edited B cells, peripheral blood cells differentiated toward PBs and PCs showed greater a-GAL activity as compared to AAV- transduced controls, indicating that integration of the GLA transgene was leading to high express! on/secreti on of a-GAL enzyme. B cells engineered to express ha-GAL both the carboxy-terminal LL truncation and the addition of a heavy chain (SEQ ID No. 3) or light chain (SEQ ID No. 4) signal peptide showed the greatest increase in a-GAL activity.
Example 3. In Vitro Expression of a-GAL in Edited Human Plasmablasts
[0174] Next, human B cells isolated from peripheral blood of five different donors (Donors A-E), differentiated towards human plasmablasts and were edited at the AAVS1 locus to express the WT ha-GAL protein (“Edited Wildtype GLA Donor A,” 1137, SEQ ID NO. 9) or the truncated version of a-GAL (“Edited WFT 2AA-trunc-GLA Donor” A through E, 1140, SEQ ID NO. 11). As a control, B cells were only transduced with rAAV6 vectors (1137 or 1140), without RNP nucleofection (unedited cells Donor A). a-GAL activity was measured using the protocol described in Example 1. As shown in FIG. 4, edited B cells/plasmablasts showed significantly enhanced a-GAL activity compared to unedited cells.
Example 4. Alpha-Galactosidase Activity in Supernatants from Plasmablasts and Plasma cells
[0175] Next, a-GAL expression was measured in edited cells under modified conditions. For this experiment, the IFNa2b concentration was increased from 1.5 ng/ml to 15 ng/ml. Cellular density was maintained at 0.6 M/mL and cells were expanded for nine days instead of seven. As shown in FIG. 5, under these conditions, edited peripheral blood cells differentiated toward PBs and PCs showed comparable secretion of active a-GAL when edited with a truncated version of a-GAL (“Edited GLA (1140)").
Example 5. Active a-GAL in Edited Human Plasmablasts.
[0176] Next, human B cells isolated from peripheral blood, differentiated towards human plasmablasts and were edited at the AAVS1 locus to again express various a-GAL forms (1140 = GLA with LL truncation and heavy chain signal peptide (SEQ ID No. 11); 1159 = GLA with LL truncation and light chain signal peptide (SEQ ID No. 12); 1501= GLA with 4 amino acids truncation at the C-terminal and heavy chain signal peptide; 1509 (SEQ ID No. 16 (GLA nt sequence), 18 (construct)), 1510 (SEQ ID No. 19), 1511 (SEQ ID No. 23) = 3 different codon optimization of GLA sequence with LL truncation and heavy chain signal peptide). As a control, B cells were only transduced with rAAV6 vectors, without RNP nucleofection (“AAV”). a-GAL activity was measured using the protocol described in Example 1. As shown in FIG. 6, edited B cells/plasmablast cells showed increased a-GAL activity when compared with AAV controls. Further, the codon optimized constructs encoding GLA with the LL truncation and either the heavy or light chain signal peptide (1509, 1510) showed improved a-GAL activity when compared with B cells/plasmablasts edited with the corresponding non-codon optimized sequences (1140, 1159). Further codon optimization studies are shown at FIG. 20 in two donors: donor 1 (FIG. 20 A) and donor 2 (FIG. 20B). When compared with episomal (AAV) expression, B cells edited to express human GLA (labeled in FIG.2 as “GLA-Edited”, and labeled in FIGs. 20A and 20B with “Edit” followed by the construct number) showed significantly enhanced a-GAL activity. Construct 1482 is the original cDNA sequence (SEQ ID NO. 4). Of the five codon optimized constructs (1588 (SEQ ID No. 26), 1598 (SEQ ID No. 27), 1599 (SEQ ID No. 28), 1600 (SEQ ID No. 29) and 1606 (SEQ ID No. 25)), construct 1606 and 1599 showed enhanced a- GAL activity in B cells/PB cells when compared to activated B cells and compared to construct 1482.
Example 6. Restoration of Intracellular a-GAL levels in Fabry Patient Fibroblasts
[0177] Fabry’s disease is characterized by a deficiency in the body’s ability to produce a- GAL enzyme. In healthy cells, phosphorylated a-GAL binds to M6P receptors (M6P-R) and is internalized in the lysosome. See, e.g., FIG. 7A. In this experiment, fibroblasts from patients with Fabry Disease were isolated and grown as primary cell culture. Fibroblasts were then co-cultured with the supernatant from HEK 293 expressing/secreting either WT a- GAL or a-GAL with both the carboxy -terminal LL truncation and the addition of a heavy chain signal peptide (SEQ ID No. 3).
[0178] Results. As shown in FIG. 7B, WT fibroblasts express ha-GAL whereas fibroblasts from Fabry patients express no ha-GAL. When fibroblasts from Fabry patients were co-cultured with supernatant from HEK 293 cells expressing/secreting a-GAL, there was a significant increase (3-fold higher) in a-GAL activity. Both a-GAL forms tested are capable of being internalized and restored intracellular a-GAL activity in Fabry patient- derived fibroblasts.
Example 7. Expression of a-GAL Protein for Fabry from Edited B cells in vivo
[0179] a-GAL activity and expression of ha-GAL protein from edited B cells was examined in vivo. Mice were injected with 30 x 106 of either un-edited or edited, activated B cells along with 3 x 106 CD4+ T cells. Engraftment and survival of human B cells in NSG is poor given the lack of additional human immune components and cytokines. CD4+ T cells play an important role in survival and function of B cells. It has been demonstrated that the co-transfer of CD4+ T cells supports survival and function of human B cells in NSG mice. See, e.g., Ishikawa, Y., et al., 2014, Eur. J. Immunol., 44:3453-3463; Luo B. et al., 2020, Cell Death and Disease, 11 :973. a-GAL activity (FIG. 9A) was measured over 102 days and protein expression (FIG. 9B) was measured over the course of 22 days.
[0180] Adoptive Transfer of Edited B Cells. Human B cells gene edited and cultured as described in Example 1 were harvested at D6 after editing and washed in PBS. Subsequently, B cells were resuspended at 30 xlO6 cells/300 ul and transferred into NSG mice via intravenous tail vein injection. Five days prior to B cells transfer, a total of 3 xlO6 CD4+ T cells were transferred into the NSG mice using the same procedure.
[0181] Isolation of Plasma from NSGMice. Mice were exposed to a heat lamp to induce vasodilation. Once the lateral tail veins were visibly dilated, the mouse were restrained and a snip at the tip of the tail was performed. 80 - 90 pL of blood were collected into an EDTA spiked plasma tube. Collected blood samples were kept on ice until processing. Subsequently, blood samples were spun at 8,000 RPM for 5 minutes and ~45- 50 pl of plasma were collected and kept frozen at -20°C until analyzed for a-GAL activity levels. a-GAL activity was measured from isolated plasma as described in Example 1 above.
[0182] Measuring a-Gal Protein. a-GAL specific activity in plasma of NSG mice was measured using the Alpha Galactosidase Activity Assay kit according to manufacturer’s instruction (Abeam, ab239716). a-GAL protein concentration was assessed using Human GLA/ Alpha Galactosidase (Sandwich ELISA) (LS Bio, LS-F 10765).
Example 8. Assessment of sgRNA efficiency and specificity and effect of codon optimization on a-GAL expression.
[0183] Described herein is a highly efficient, specific and safe gene editing strategy targeting an AAV1 locus. Healthy donor B cells are isolated from peripheral blood and engineered to expresses a-GAL, using CRISPR/Cas-based genome editing using the methods described above. Engineered B cells were expanded and cultured using the experimental methods described above and as set forth in FIG. 13A and FIG. 13B. Genomic DNA was extracted to measure efficiency of site-specific mutations (FIG. 9A) and targeted integration frequency, using digital PCR assays (n= 3 healthy donors) (FIG. 9B).
[0184] Human B cells were edited using the techniques described in example 1 and using various GLA-cDNA sequences (FIG. 10A (SEQ ID NO. 14) and FIG. 10B (SEQ ID NO. 24) were cultured for 12 days. At day 9 and 12, cell culture supernatants were collected, cell counts and viability were assessed. Functional, secreted a-GAL was quantified in supernatants using an activity assay and results were normalized to cell counts.
[0185] RNPs targeting an AAV1 locus were delivered into B cells via nucleofection and genomic DNA was extracted to measure efficiency of site-specific mutations using digital PCR assays sequences (FIG. 11 A (SEQ ID NO. 11) and FIG. 1 IB (SEQ ID NO. 25)) n=3 healthy donors).
[0186] Guide-SEQ analysis of genomic DNA isolated from human B cells treated with AAV1 specific RNPs. On-target/Off-target sites were identified by incorporation of doublestranded oligodeoxynucleotides (dsODNs) (FIG. 12).
Example 9. Assessment of B Cell Lineages After In Vitro Conditioning.
[0187] B Cell phenotype was assessed in engineered B cells after in vitro conditioning.
[0188] Flow Cytometry. Phenotypic analysis of B cells was performed by flow cytometry. Cells were stained in Cell Staining Buffer (Biolegend) and Human TruStain FcX™ (Fc Receptor Blocking Solution; Biolegend) was used to avoid FcRs-mediated nonspecific antibody binding. Cells were stained with the following antibody- fluorochrome combination: IgD- AF488 (Biolegend), CD20- BV711 (Biolegend), CD38-PE (Biolegend), CD27-BV605 (Biolegend), CD138-BV421 (Biolegend).
[0189] It was found that ex-vivo conditioning promotes B cell differentiation toward a plasma cell-like phenotype, expressing high levels of engineered a-GAL. B cells lineages after in vitro conditioning. Memory B cells (CD27+IgD-), Plasmablasts (PBs) (CD27+CD38+ CD20-), Plasma cells (PCs) (CD27+CD38+ CD20- CD 138+ populations were monitored using Flow cytometry (FIG. 14A). PrimeFlow analysis of GLA transcripts in B cells showed that the CD27+CD38+ CD20- cell population express higher levels of GLA transcripts than the CD27- cell population (FIG. 14B).
Example 10. In Vivo Adoptive Transfer of Genetically Engineered B Cells.
[0190] a-GAL activity protein from edited B cells was examined in vivo. Mice were injected with 30 x 106 of either un-edited or edited, activated B cells along with 3 x 106 CD4+ T cells as described in Example 7 above. Human B cells gene edited and cultured as described in Example 1 were harvested at D6 after editing and washed in PBS. Subsequently, B cells were resuspended at 30 xlO6 cells/300 ul and transferred into NSG mice via intravenous tail vein injection. Five days prior B cells transfer total 3 xlO6 CD4+ T cells were transferred into the NSG mice using the same procedure. Plasma was isolated from NGS mice as described in Example 7 above and a-GAL activity was measured from isolated plasma as described in Example 1 above.
[0191] Circulating a-GAL levels were monitored every 7-10 days via an enzymatic activity assay on plasma samples (n= 2 healthy donors). Engineered B Cells engrafted in
NSG mice produced sustained, supraphysiologic a-GAL plasma levels (FIG. 15). Luciferase imaging showed early engraftment of engineered cells in bone marrow (FIG. 16A).
Engrafted B cells were isolated from multiple murine tissues and the cell phenotype was assessed using Flow cytometry (n=4 mice) using the methods described in Example 8 (FIG. 16B).
Example 11. Reduction in GvHD symptoms by infusion of memory CD4+ cells.
[0192] B cell engraftment in NSG mice is extremely poor in the absence of support from additional human immune components or cytokines (Luo 2020; Cheng 2022). NSG mice humanized via adoptive transfer of autologous total human CD4+ T cells prior or together with human B cells dramatically increased the ability of B cells to engraft and survive in vivo (Levy 2016).
[0193] One of the challenges in developing humanized mouse models using T cells to
“humanize” the host is development of graft versus host disease (GvHD), which limits the study period as mice must be sacrificed for ethical reasons. It has been shown that adoptive transfer of memory CD4+ T cells (CD4+CD45RO+) in NSG mice dramatically reduced GvHD onset and severity (Anderson 2003) while supporting human B cells engraftment and function (Ishikawa 2014).
[0194] In another, in vivo adoptive transfer study, memory CD4+ T cells (CD4+CD45RO+) were to reduce the incidence and delay the onset of GvHD. a-GAL activity and expression of ha-GAL protein from edited B cells was examined in vivo. Mice were injected with 25 x 106 of either un-edited or edited, activated B cells along with 3 x 106 CD4+ T cells as described in Example 7 above. Human B cells gene edited and cultured as described in Example 1 were harvested at D6 after editing and washed in PBS. Subsequently, B cells were resuspended at 25 xlO6 cells/300 pl and transferred into NSG mice via intravenous tail vein injection. Five days prior B cells transfer total 3 xlO6 CD4+ T cells were transferred into the NSG mice using the same procedure. Plasma was isolated from NGS mice as described in Example 7 above and a-GAL activity was measured from isolated plasma as described in Example 1 above.
[0195] Circulating a-GAL levels were monitored every 7-10 days via an enzymatic activity assay on plasma samples. Engineered B Cells engrafted in NSG mice produced sustained, supraphysiologic a-GAL plasma levels (FIG. 18). NSG mice were humanized as
described in FIG 18 were assessed for bodyweight and GVHD symptoms (e.g., hair loss, redness ears/extremities, hunched posture). Compared with saline treated mice (FIG. 19A), mice infused with Memory CD4+ T cells together with edited B cells (1140, SEQ ID NO. 11) (FIG. 19B) showed no difference in body weight or GVHD symptoms as compared to saline controls. In contrast, mice infused with Total CD4+ T cells together with edited B cells (1140, SEQ ID NO. 11) showed reduction in body weight and/or exhibited GVHD symptoms. (FIG. 19C).
Example 12. Gene editing of a functional GLA cDNA in Fabry patient B cells
[0196] Next, human B cells isolated from peripheral blood of a Fabry patient activated and were edited at the AAVS1 locus to again express construct 1606 (SEQ ID NO. 25). B cells were isolated from Fabry patient’s PBMCs via CD19+ positive selection, and activated for 3 days in media containing CD40L, CpG, IL-2, IL- 10, IL- 15 and IL-21. Gene editing was performed on day 3 after isolation using Cas9/sgRNA delivered via electroporation followed by transduction via AAV6 GLA vector (1606, SEQ ID No. 25). Engineered B cells were expanded in the same media describe above for additional 7 days.
[0197] Edited Fabry B cells we able to expand in vitro over time (FIG. 17A). Edited Fabry B cells demonstrated on-target integration of approximately 60 copies per 100 cells, as measured by digital PCR (FIG. 17B). Finally, edited Fabry B cells secreted active a-GAL enzyme, where unedited cells did not (FIG. 17C).
Claims
CLAIMS A therapeutic protein comprising a modified human a-GAL protein, wherein the modified human a-GAL protein has been modified by, a. a deletion of two carboxy terminal leucine residues of a wild-type human a- GAL protein; and b. a replacement of a signal peptide of said wild-type human a-GAL protein with an immunoglobulin(Ig) signal peptide. The therapeutic protein of claim 1, wherein the signal peptide is a heavy chain Ig signal peptide. The therapeutic protein of claim 1, wherein the signal peptide is a light chain Ig signal peptide. The therapeutic protein of claim 1, wherein the therapeutic protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. The therapeutic protein of claim 1, wherein the therapeutic protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. The therapeutic protein of claim 1, wherein the therapeutic protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. The therapeutic protein of claim 1, wherein the therapeutic protein comprises an amino acid sequence that is selected from: SEQ ID Nos: 2-4. The therapeutic protein of claim 1, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. The therapeutic protein of claim 1, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21.
The therapeutic protein of claim 1, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. The therapeutic protein of claim 1 , wherein the modified human a-GAL protein is encoded by a nucleic acid sequence is selected from: SEQ ID Nos: 6-8, 16-17 or 20- 21. An engineered human B cell comprising a therapeutic protein, wherein the therapeutic protein comprises a modified human GLA wherein the modified human a-GAL protein has been modified by, a. a deletion of two carboxy terminal leucine residues of a wild-type human a- GAL protein; and b. a replacement of a signal peptide of said wild-type human a-GAL protein with an immunoglobulin(Ig) signal peptide. The engineered B cell of claim 12, wherein the engineered B cell further comprises a nucleic acid sequence encoding said therapeutic protein, and wherein said nucleic acid sequence has been inserted into either the AAVS1 or the human ROSA26 locus. The engineered B cell of claim 12, wherein the signal peptide is a heavy chain Ig signal peptide. The engineered B cell of claim 12, wherein the signal peptide is a light chain Ig signal peptide. The engineered B cell of claim 12, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 85% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. The engineered B cell of claim 12, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 90% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4.
The engineered B cell of claim 12, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 95% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. The engineered B cell of claim 12, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is selected from: SEQ ID Nos: 2-4. The engineered B cell of claim 12, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. The engineered B cell of claim 12, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. The engineered B cell of claim 12, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. The engineered B cell of claim 12, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. The engineered B cell of any one of claims 12-22, wherein the gene has been inserted into the human AAVS1 locus. The engineered B cell of any one of claims 12-22, wherein the gene has been inserted into the human ROSA26 locus. A population of cells comprising a plurality of engineered B cells of any one of claims 12-22, wherein at least 20% of the population of cells express said therapeutic protein. The engineered B cell of any one of claims 12-22, wherein said B cell is CD20+. The engineered B cell of any one of claims 12-22, wherein said B cell is CD20-.
A population of cells comprising a plurality of engineered B cells according to any one of claims 12-22, wherein a majority of said cells are CD20+. A population of cells comprising a plurality of engineered B cells according to any one of claims 12-22, wherein more than 80 percent of said cells are CD20-. The engineered B cell of any one of claims 12-22, wherein said B cell is a plasmablast. A population of cells comprising a plurality of engineered B cells according to any of any one of claims 12-22, wherein a majority of said cells are plasmablasts. The engineered B cell of any one of claims 12-22, wherein said cell is a plasma cell. A population of cells comprising a plurality of engineered B cells according to any one of claims 12-22, wherein a majority of said cells are plasma cells. A method of producing an engineered B cell expressing a therapeutic protein, the method comprising delivering to a human B cell: a. an RNA-guided nuclease b. a gRNA targeting a locus on the human genome; and c. a construct comprising a nucleic acid sequence encoding a therapeutic protein, wherein the therapeutic protein comprises a modified human a-GAL protein. The method of claim 35, wherein the RNA-guided nuclease and gRNA are delivered to the B cell as an RNP. The method of claim 35, wherein the RNA-guided nuclease and gRNA are delivered to the B cell as a nanoparticle. The method of claim 35, wherein the RNA-guided nuclease and gRNA are delivered to the B cell via electroporation. The method of claim 35, wherein the construct delivered to the B cell using a viral vector. The method of claim 35, wherein the construct delivered to the B cell as double stranded DNA. The method of claim 35, wherein the RNA-guided nuclease comprises the amino acid sequence of SEQ ID No. 14 or SEQ ID No. 24.
The method of claim 35, wherein the RNA-guided nuclease comprises the amino acid sequence of SEQ ID NO. 15. The method of claim 35, wherein the targeting construct comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 9-13 18-19 and 25-29. The method of any one of claims 35-43, wherein said engineered B cell is CD20-. The method of any one of claims 35-43, wherein said engineered B cell is CD20+. A population of cells comprising a plurality of engineered B cells according to any one of claims 35-43, wherein a majority of said cells are CD20+. A population of cells comprising a plurality of engineered B cells according to any one of claims 35-43, wherein more than 80 percent of said cells are CD20-. The method of any one of claims 35-43, wherein said engineered B cell is a plasmablast. A population of cells comprising a plurality of engineered B cells according to any of any one of claims 35-43, wherein a majority of said cells are plasmablasts. The method of any one of claims 35-43, wherein said engineered B cell is a plasma cells. A population of cells comprising a plurality of engineered B cells according to any one of claims 35-43, wherein a majority of said cells are plasma cells. The method of any one of claims 35-45, wherein the population of cells comprises either plasmablasts, plasma cells or both plasmablasts and plasma cells. A method of producing an engineered B cell expressing a therapeutic protein, the method comprising delivering to a human B cell: a. a RNA-guided nuclease; b. a gRNA targeting the AAVS1 gene, wherein the gRNA comprises the nucleic acid sequence of SEQ ID NO. 14, 15 or 24; and c. a construct comprising a nucleic acid sequence encoding a therapeutic protein and a left and right homology arm; wherein the construct comprises a nucleic
acid sequence selected from the group consisting of SEQ ID NOs. 9-13 and 18-19, wherein said engineered B cell expresses said therapeutic protein. A method of treating a patient in need thereof comprising administering to said patient an engineered human B cell, wherein the engineered human B cell has been edited to express a therapeutic protein encoding a human a-GAL protein. The method of claim 54, wherein the therapeutic protein comprises a modified human GLA wherein the modified human a-GAL protein has been modified by, a. a deletion of two carboxy terminal leucine residues of a wild-type human a- GAL protein; and b. a replacement of a signal peptide of said wild-type human a-GAL protein with an immunoglobulin(Ig) signal peptide. The method of claim 55, wherein the signal peptide is a heavy chain Ig signal peptide. The method of claim 55, wherein the signal peptide is a light chain Ig signal peptide. The method of claim 55, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 85% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. The method of claim 55, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 90% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. The method of claim 55, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 95% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. The method of claim 55, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is selected from: SEQ ID Nos: 2-4. The method of claim 55, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21.
The method of claim 55, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. The method of claim 55, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. The method of claim 55, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. The method of any one of claims 55-65, wherein the gene encoding the therapeutic protein has been inserted into the human AAVS1 locus. The method of any one of claims 55-65, wherein the gene encoding the therapeutic protein has been inserted into the human ROSA26 locus. The method of any one of claims 55-65, wherein at least 20% of the population of cells express said therapeutic protein. The method of any one of claims 55-65, wherein said population of cells comprise cells that are CD20+. The method of any one of claims 55-65, wherein said population of cells comprise cells that are CD20-. A population of cells comprising a plurality of engineered B cells according to any one of claims 55-65, wherein a majority of said cells are CD20+. A population of cells comprising a plurality of engineered B cells according to any one of claims 55-65, wherein more than 80 percent of said cells are CD20-. The method of any one of claims 55-65, wherein said engineered B cell is a plasmablast. A population of cells comprising a plurality of engineered B cells according to any of any one of claims 55-65, wherein a majority of said cells are plasmablasts.
The method of any one of claims 55-65, wherein said engineered B cell is a plasma cells. A population of cells comprising a plurality of engineered B cells according to any one of claims 55-65, wherein a majority of said cells are plasma cells. The method of any one of claims 55-65, wherein the population of cells comprises either plasmablasts, plasma cells or both plasmablasts and plasma cells. A method of treating a patient in need thereof comprising administering to said patient an engineered human B cell, wherein said human B cell comprises a therapeutic protein, encoded by a gene, wherein the therapeutic protein comprises a modified human a-GAL protein, wherein the modified human a-GAL protein has been modified by, a. a deletion of two carboxy terminal leucine residues of a wild-type human a- GAL protein; and b. a replacement of a signal peptide of said wild-type human a-GAL protein with an immunoglobulin(Ig) signal peptide. The method of claim 78, wherein the nucleic acid sequence encoding said therapeutic protein has been inserted into either the AAVS1 or the human ROSA26 locus. The method of claim 78, wherein the signal peptide is a heavy chain Ig signal peptide. The method of claim 78, wherein the signal peptide is a light chain Ig signal peptide. The method of claim 78, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 85% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. The method of claim 78, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 90% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. The method of claim 78, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 95% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4.
The method of claim 78, wherein the modified human a-GAL protein comprises an amino acid sequence that is selected from: SEQ ID Nos: 2-4. The method of claim 78, w wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. The method of claim 78, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. The method of claim 78, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. The method of claim 78, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. The method of any one of claims 78-89, wherein the gene has been inserted into the human AAVS1 locus. The method of any one of claims 78-89, wherein the gene has been inserted into the human ROSA26 locus. The method of claim 78-89, wherein at least 20% of the population of engineered B cells, express said therapeutic protein. The method of any one of claims 78-89, wherein said population of cells comprise cells that are CD20+. The method of any one of claims 78-89, wherein said population of cells comprise cells that are CD20-. A population of cells comprising a plurality of engineered B cells according to any one of claims 78-89, wherein a majority of said cells are CD20+. A population of cells comprising a plurality of engineered B cells according to any one of claims 78-89, wherein more than 80 percent of said cells are CD20-.
The method of any one of claims 78-89, wherein said engineered B cell is a plasmablast. A population of cells comprising a plurality of engineered B cells according to any of any one of claims 78-89, wherein a majority of said cells are plasmablasts. The method of any one of claims 78-89, wherein said engineered B cell is a plasma cells. A population of cells comprising a plurality of engineered B cells according to any one of claims 78-89, wherein a majority of said cells are plasma cells. The method of any one of claims 78-89, wherein the population of cells comprises either plasmablasts, plasma cells or both plasmablasts and plasma cells. A method of treating a patient in need thereof comprising administering to said patient a therapeutic protein comprising a modified human a-GAL protein, wherein the modified human a-GAL protein has been modified by, a. a deletion of two carboxy terminal leucine residues of a wild-type human a- GAL protein; and b. a replacement of a signal peptide of said wild-type human a-GAL protein with an immunoglobulin(Ig) signal peptide. The method of claim 102, wherein the signal peptide is a heavy chain Ig signal peptide. The method of claim 102, wherein the signal peptide is a light chain Ig signal peptide. The method of claim 102, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 85% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. The method of claim 102, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 90% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4.
The method of claim 102, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 95% identical to the amino acid sequence selected from: SEQ ID Nos: 2-4. The method of claim 102, wherein the modified human a-GAL protein comprises an amino acid sequence that is selected from: SEQ ID Nos: 2-4. The method of claim 102, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. The method of claim 102, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. The method of claim 102, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence selected from: SEQ ID Nos: 6-8, 16-17 or 20-21. The method of claim 102, wherein the modified human a-GAL protein is encoded by a nucleic acid sequence that is selected from: SEQ ID Nos: 6-8, 16-17 or 20-21.
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US202263400664P | 2022-08-24 | 2022-08-24 | |
US63/400,664 | 2022-08-24 | ||
US202363484668P | 2023-02-13 | 2023-02-13 | |
US63/484,668 | 2023-02-13 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2024044697A2 true WO2024044697A2 (en) | 2024-02-29 |
WO2024044697A3 WO2024044697A3 (en) | 2024-04-04 |
Family
ID=90014093
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2023/072836 WO2024044697A2 (en) | 2022-08-24 | 2023-08-24 | Compositions and methods for treatment of fabry disease |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2024044697A2 (en) |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR100547402B1 (en) * | 1996-09-13 | 2006-02-01 | 트랜스케리오틱 쎄러피스, 인코포레이티드 | Therapy for ?-galactosidase a deficiency |
GB201508025D0 (en) * | 2015-05-11 | 2015-06-24 | Ucl Business Plc | Fabry disease gene therapy |
BR112019022473A2 (en) * | 2017-04-27 | 2020-10-20 | Immusoft Corporation | b cells for in vivo release of therapeutic agents and dosages of these |
JP2022527302A (en) * | 2019-03-28 | 2022-06-01 | インテリア セラピューティクス,インコーポレイテッド | Polynucleotides, compositions, and methods for polypeptide expression |
-
2023
- 2023-08-24 WO PCT/US2023/072836 patent/WO2024044697A2/en unknown
Also Published As
Publication number | Publication date |
---|---|
WO2024044697A3 (en) | 2024-04-04 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP7365374B2 (en) | Nuclease-mediated gene expression regulation | |
US11679130B2 (en) | Genetically engineered t cells with Regnase-1 and/or TGFBRII disruption have improved functionality and persistence | |
JP6722176B2 (en) | Methods and compositions for nuclease-mediated genomic engineering and correction in hematopoietic stem cells | |
JP2018525979A (en) | Delivery methods and compositions for nuclease-mediated genomic gene manipulation | |
CA3086187A1 (en) | Vcar compositions and methods for use | |
JP7198823B2 (en) | Genetically engineered hematopoietic stem cells as a platform for systemic protein expression | |
US20230346836A1 (en) | Genetically engineered t cells with disrupted casitas b-lineage lymphoma proto-oncogene-b (cblb) and uses thereof | |
US20210147798A1 (en) | Artificially Manipulated Immune Cell | |
JP2021520844A (en) | Manipulated cascade components and cascade complexes | |
JP2022519070A (en) | Gene regulation compositions and methods for improving immunotherapy | |
WO2022247873A1 (en) | Engineered cas12i nuclease, effector protein and use thereof | |
US20220193134A1 (en) | Co-use of lenalidomide with car-t cells | |
US11926676B2 (en) | Masked chimeric antigen receptor specific to tyrosine-protein kinase like 7 (PTK7) and immune cells expressing such | |
US20200338213A1 (en) | Systems and methods for treating hyper-igm syndrome | |
WO2024044697A2 (en) | Compositions and methods for treatment of fabry disease | |
US20230212613A1 (en) | Methods for targeted insertion of exogenous sequences in cellular genomes | |
CN115141807A (en) | Methods of treating beta-thalassemia | |
WO2022226020A2 (en) | Engineering b cell-based protein factories to treat serious diseases | |
US20220288122A1 (en) | Genetically engineered t cells with ptpn2 knockout have improved functionality and anti-tumor activity | |
WO2024062388A2 (en) | Genetically engineered immune cells expressing chimeric antigen receptor targeting cd20 | |
IL302315A (en) | Safe harbor loci |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 23858309 Country of ref document: EP Kind code of ref document: A2 |