CN108795946B - Recombinant adeno-associated virus carrying SMN1 gene expression cassette and application thereof - Google Patents

Abstract

The invention provides a series of recombinant adeno-associated viruses carrying artificially designed SMN1 gene expression cassettes. In vivo experiments show that the recombinant adeno-associated virus vector can be efficiently introduced into the central nervous system, continuously and stably express the SMN1 protein, prolong the life cycle of a Spinal Muscular Atrophy (SMA) model animal, increase the weight of the SMA model animal and restore the growth and development of the SMA model animal. The result indicates that the recombinant adeno-associated virus vector can be developed into a novel drug for treating spinal muscular atrophy caused by SMN1 gene mutation.

Description

Recombinant adeno-associated virus carrying SMN1 gene expression cassette and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an artificially designed SMN1 gene expression cassette carried by a recombinant adeno-associated virus vector and application thereof in treating spinal muscular atrophy.

Background

Spinal Muscular Atrophy (SMA) is a common group of autosomal recessive genetic diseases in children and adolescents. Foreign literature suggests that the incidence rates are between 1/6000 and 1/10000(Nicole S, et al, Muscle & nerve 2002;26(1):4-13.) and the carrier frequency in the population is between about 1/40 and 1/50. The incidence of the southern population in China is estimated to be 1/53000(Chung B, et al J Child neuron 2003; 18(3): 217-219.).

The disease is characterized by muscular atrophy and paralysis caused by degeneration of anterior motor neurons of the spinal cord (Maryam Oskoui, et al. neuro therapeutics, 2008; 5: 499-. If intercostal muscles are affected, respiratory function is also affected, endangering life. The survival rate of patients generally depends on whether respiratory function is affected, and motor-related muscle groups tend to reduce the quality of life of patients, but are not life-threatening (Thomas TH, et al, neurological disorders, 1994; 4(5-6): 497-. There are some patients who also have finger fibrillation (Iannaclone ST. sensors in neurology, 1998; 18(1): 19-26.). Facial muscles are generally rarely affected (Iannacsone ST, et al, Pediatric neurology, 1993; 9(3): 187. sup. 193.). In SMA patients, the cognitive function is generally normal, and in the later stages of SMA disease, severe separation of mental and motor abilities often occurs (Thomas TH, et al, neurological disorders, 1994; 4(5-6): 497-.

The international association for SMA establishes the classification rules of SMA according to the age, motor ability and life span of the patients. SMA is classified into 3 types, i.e., type I, type II and type III (Munsat T. Neurousaral disorders, 1991; 1(2):81. Munsat TL, et al. Neurousaral disorders, 1992; 2(5-6): 423-.

Spinal muscular atrophy type I is the most common type (also known as Werdnig-hoffmann disease, soma SMA, infancy SMA). Generally, the disease occurs within 6 months after birth. Children with SMA type I are generally unable to sit alone and die most before two years of age (Cobben JM, et al, neurousaral disorders, 2008; 18(7): 541-. This type is characterized by severe progressive muscle weakness and muscle tone weakness (Iannacsone ST. sensines in neurology, 1998; 18(1): 19-26.). Type I SMA is one of the most important genetic diseases causing infant death (Nicole S, et al, Muscle & nerve 2002;26(1): 4-13.).

SMA type II is intermediate (also known as juvenile SMA, chronic SMA). The disease is developed in 6-18 months. Such patients may sit alone, but may not walk on their own. And respiratory dysfunction frequently occurs (Bertini E, et al. Neurousaral disorders. 2005;15(11): 802-. Typically such patients have a lifespan of more than two years, and sometimes may even survive to puberty, or even longer (Zerres K, et al. Journal of the Neurological sciences. 1997; 146(1): 67-72.).

SMA type III is light SMA (also known as Kugelberg-Welander disease, Wohlfart-Kugelberg-Welander disease). Generally, the disease occurs after 18 months, some patients can walk independently, and the puberty disease condition is likely to repeat. The lifespan is mostly normal (Zerres K, et al, Journal of the Neurological sciences, 1997; 146(1): 67-72.).

There is a reported severe form of SMA, which begins to develop before birth and dies in the first month, called SMA0, also known as congenital SMA (Dubowitz V. European Journal of Pediatric neuroy 1999; 3(2): 49-51.). The other extreme in the spectrum of SMA disease is type SMA IV, also known as adult SMA, which typically develops after age 35 (Maryam Oskoui, et al, neuro therapeutics, 2008; 5:499- & 506.).

SMA has a broad clinical spectrum in nature, with each subtype under the three major types, and there is also overlap between the subtypes. For example, some children have a long life span although they cannot sit alone (Thomas TH, et al, Neurousavular disorders, 1994; 4(5-6): 497-502.). Some children patients can move their heads autonomously although they belong to SMA I type (the characteristic expression of SMA I type means that the heads cannot move autonomously); after some SMA I type children suffer from the disease, the disease does not progress rapidly, and some children suffer from the disease before 6 months but can sit alone; some develop within 18 months, but can be isolated (Russman BS, et al J Child neuro 1992; 7(4): 347-. It has therefore been proposed that the maximal exercise capacity exhibited by the infant after onset of disease is better than the onset time to predict the severity of the disease in the infant (Zerres K, et al, neuro-temporal disorders 1999; 9(4): 272-.

The SMA virulence gene, which maps to 5q13.3, was designated the SMN (motoneuron survival gene) gene (Brzustowicz LM, et al Nature 1990; 344(6266): 540-. The SMN gene is 20kb in length, the cDNA is 1.7kb, and 8 exons are contained. The gene has 2 forms, SMN1 (SMNt) near telomeres and SMN2 (SMNc) near centromeres (Nicole S, et al, Muscle & nerve. 2002;26(1): 4-13.). Both are highly homologous (99%), differ in coding region only by a synonymous mutation in exon 7 (codon 280, SMN1 TTC, SMN2 TTT), and all other differences are located in non-coding regions, without affecting coding function (Nicole S, et al, Muscle & nerve. 2002;26(1): 4-13.).

SMN encodes the full-length SMN transcript fl-SMN (full-length motor neuron transcript) encodes 1 protein of 294 amino acids with a molecular weight of 38kD, referred to as SMN protein. SMN proteins are widely expressed in tissues and cells of all mammalian organisms, with high levels in the brain, kidney, liver, especially motor neurons, moderate levels in skeletal muscle and cardiac muscle cells, and low levels in lymphocytes. The expression of SMN protein in motor neurons was found to be stable at all ages, while the amount of SMN protein in other tissues and organs decreased with age, indicating that the survival and function of motor neurons strictly depend on high levels of SMN protein expression (Wu Shi, et al, J. Chinese genetics. 2003; 20(5): 430-432.).

The specific function of SMN proteins is currently unknown (Sumner CJ. J Child neuro. 2007; 22(8): 979-. In motor neurons, splicing of mRNA is likely to be dependent on the SMN protein (Lefebvre S, et al cell. 1995; 80(1): 155-. The motor neuron survivin may also play a key role in The growth of neuronal axons (McWhorter ML, et al, The Journal of Cell biology 2003; 162(5): 919-. However, a reduction in SMN protein was found in all cells of SMA patients. It is unknown whether the absence of this protein has an effect only on the function of motor neurons (Zou T, et al, Biochemical and Biophysical Research communication 2007; 364(4): 850-. There are also a number of other hypotheses for the pathological process of SMA, including deregulation of the apoptotic program, excitotoxicity of glutamate production, oxidative stress, and so forth (Takeuchi Y, et al. J Child neuro 1994; 9(3): 287 and 289).

SMN proteins are distributed predominantly in the cytoplasm in the central nervous system and in the nucleus in skeletal muscle, and aggregate to a gems structure in the nucleus (Patrizi AL, et AL Eur J Hum Gene 1999; 7(3): 301-. The causes of SMA are that the expression level of SMN, a functional protein, is reduced due to homozygous deletion or compound heterozygous mutation of the SMN1 gene, so that motor neuron cells at anterior horn of spinal cord are degenerated, and muscle atrophy and paralysis are caused (Wu Shi, et al, J. Chinese genetics 2003; 20(5): 430-.

The expression product of the SMN1 gene is 90% SMN protein, while the SMN2 gene transcript is 90% absent of exon 7, only 10% of transcripts containing exon 7 express only about 10% of SMN protein (Lorson Cl, et al. Proceedings of the National Academy of Sciences of the United States of America 1999; 96(11): 6307-. The aberrant splicing of SMN2 resulted in a transcript lacking exon 7, termed Δ 7SMN 2. There are two main views of the mechanism by which Δ 7SMN2 is generated: cartegni et al believe that the mutation of the +6 base C to T occurring at exon 7 of the SMN2 gene disrupts the Exon Splicing Enhancer (ESE), resulting in the splicing of exon 7 of SMN2 (Cartegni L, et al, American Journal of Human genetics 2006; 78(1): 63-77.); kashima and Manley found that mutation of C into T resulted in an Exon Splicing Silencer (ESS) that could recruit hnRNP A1 inhibitor protein to SMN2, resulting in cleavage of SMN2 exon 7 (Tsuyoshi Kashima, et al. Nature genetics. 2003; 34: 460. sup. 463.). It was demonstrated that the alteration of another oligonucleotide at position +100 of intron 7 resulted in a binding site for hnRNP A1 at SMN2 which is essential for exon 7 splicing, and that mutations which blocked the binding of this protein increased the production of fl-SMN RNA containing exon 7 (Tsuyoshi Kashima, et al. PNAS. 2007; 104(9): 3426-.

In addition, the self-oligomerization of the SMN protein is important for its function (Wolstencroft EC, et al. Hum Mol Genet 2005; 14: 1199-. While the truncated Δ 7SMN2 protein mainly produced by SMN2 gene expression has reduced self-oligomerization capability, can improve SMA in vitro but cannot prevent the SMA phenotype, is biochemically unstable, is degraded quickly, and cannot compensate the deletion of SMN1 (Le TT, et al Hum Mol Genet 2005; 14: 845).

In conclusion, SMNl is defective and the normal SMN2 gene is not fully compensated, and as a result, SMN protein deficiency is a major pathogenesis of SMA. Therefore, adopting various approaches to improve SMN protein expression is the most fundamental and promising therapeutic strategy for SMA. The SMN-dependent therapeutic strategies mainly comprise SMNl gene replacement therapy, enhancement of the activity of a SMN2 gene promoter, increase of the expression of a full-length transcript of an SMN2 gene, stabilization of SMN and the like. Specific treatment regimens broadly include 3 classes: gene therapy, antisense oligonucleotides, small molecule compounds. In addition, there are therapeutic strategies that do not rely on the elevation of SMN proteins, such as stem cell therapy, neuromuscular protective drugs, and the like. Currently, 33 studies of at least 18 SMA drug candidates have entered clinical trials for the different treatment regimens described above (http:// clinicaltirials. gov).

SMN-dependent therapy: (1) gene therapy: the gene therapy method is to introduce exogenous SMN gene via virus vector to increase in vivo SMN protein expression directly, and is one promising candidate SMA therapy method. Foust et al 2009 reported that type 9 self-complementing adeno-associated virus vectors (scAAV 9) pass through the blood brain barrier more easily than other serotype adeno-associated virus vectors, can be transported to the central nervous system after intravenous injection, and infect about 60% of motor neurons (Foust KD, et al Nat Biotechnol. 2009; 27(1): 59-65.), which provides a new idea for the treatment of SMA. Subsequently, various scAAV9(scAAV9-SMN) carrying SMN1 gene expression cassettes were beginning to be used more extensively in SMA treatment studies. Foust et al established heavy type delta 7SMA mouse model (hereinafter referred to as "delta 7SMA mouse"), recombinant 9 type self-complementary adeno-associated virus vector scAAV9-CB-SMN1 of SMN1 gene expression frame carrying CB promoter (composed of human cytomegalovirus enhancer and chicken beta-actin promoter) was expressed in 5 × 10¹¹The dose of vg (viral genome)/na (t) was injected via facial vein into the mice model of heavy-duty Δ 7SMA the day after birth, and the survival was extended to 250 d, which was significantly longer than the mean survival of Δ 7SMA mice in the non-administered group by 15.5 d (Foust KD, et al Nat Biotechnol. 2010; 28(3): 271-274.). In addition, the weight, motor function and pathological manifestations of the mice in the administration group are all obviously superior to those of the mice in the non-administration group. Valori et al designed a scAAV9 virus with a gene expression cassette different from that of scAAV9-CB-SMN 1. The virus employs human cytomegalovirus promoter (CMV) regulatory sequences to optimize the expression cassette of the SMN1 gene. Mixing the virus at 1 × 10¹¹The dose of vg (viral genome)/vg (Viral genome) was injected via the facial vein into Δ 7SMA mice after the first day of birth, and the results showed that the mice body weight, motor ability and pathological manifestations were improved to different extents with an average extension of 69 days (Valori CF, et al Science comparative medicine 2010; 2(35):35ra 42.). Dominguez et al use phosphoglycerate Promoter (PGK) to regulate and optimize SMN1 gene expression, and scAAV9 to carry and construct SMN1 expression cassette, 4.5 × 10¹⁰vg（viral genome)/mouse dose was injected via the temporal vein into Δ 7SMA mice on day one after birth, and the survival time of model mice was extended to 160 days (mean value), and the weight and exercise capacity of mice were also increased (domiguez E, et al. Human Molecular genetics. 2011; 20(4): 681-693.). Three different SMN1 expression frame structures are designed in the three researches, and the SMN1 expression frame structures can be effectively introduced into a central nervous system after being carried by the scAAV9 vector to express the SMN1 protein and show a therapeutic effect. The role of scAAV9-CB-SMN1 was also demonstrated in other mammalian models (Duque SI, et al Ann neurol. 2015; 77(3): 399-. The results of an SMA pig model experiment carried out by Duque et al show that before the occurrence of SMA symptoms, the gene therapy of scAAV9-CB-SM 1 can prevent the decline of the muscle strength and the abnormal change of electromyogram of piglets; after the SMA piglet had symptoms such as hind limb muscle strength decline and gait abnormality, the gene therapy of scAAV9-CB-SMN1 was adopted, but the hind limb muscle weakness symptom was not completely improved, but was not further aggravated or completely paralyzed, which proved that the gene therapy after the symptoms appeared could still prevent the further aggravation of SMA symptoms (Duque SI, et al. Ann neurol. 2015; 77(3):399 one 414.). Meyer et al found that intracerebroventricular administration of scAAV9-CB-SMN1 reduced the effective dose by nearly 10-fold in both mouse and monkey in vivo experiments (Meyer K, et al. Mol. The 2015; 23(3): 477-487.). Based on the appreciable efficacy of this treatment regimen in animal models, the united states Food and Drug Administration (FDA) has approved scAAV9-CB-SMN1 (AVXS-101) into phase I clinical trial phase 2014, with 15 children suffering from SMAl type. The results reported in the New England journal of medicine in 2017 show that 15 patients with SMA1 type all have enhanced motor function and prolonged life cycle after receiving AVXS-101 treatment (Mendell JR, et al. N Engl J Med. 2017; 377: 1713-. Currently, the observation of 15 subjects is still ongoing.

(2) Antisense oligonucleotides (antisense oligonucleotides, ASOs): the artificially synthesized ASOs segments are specifically combined with the splicing inhibitory sequence of the exon 7 of the SMN2 gene according to Watson-Crick base pairing rule to promoteCorrect splicing of exon 7 to promote expression of full-length SMN2 transcript. In ASOs currently used in the study of SMA treatment, the effect of binding to intron 7 splicing silencer Nl sequence (ISS-N1) of SMN2 Gene is more remarkable (Singh NK, et al Mol Cell biol. 2006; 26(4):1333-1346, Hua YM, et al PLoS biol. 2007; 5(4): e73, Zhou HY, et al Hum Gene ther. 2013; 24(3): 331-342.15-17). Several cell models and mouse model experiments that have been performed internationally demonstrate that 2' -O-Methyl (MOE) ASO and morpholine ASO can increase full-length SMN2 gene expression, raise SMN protein levels, and improve SMA-associated performance (Mitrpent C, et al, PLoS one. 2013; 8(4): e62114. Keil JM, et al Mol Ther Nucleic acids. 2014; 3: e174. Osman EY, et al. Hum Mol Genet. 2014; 23(18): 4832-. After single injection of morpholine ASO into the lateral ventricles, Zhou et al up-regulate the expression of full-length SMN2 transcripts in the brain and spinal cord of mice, and prolong the survival time to 230 days, which is obviously longer than that of untreated group by about 9 days (Zhou HY, et al Hum Gene ther, 2013; 24(3): 331-342.17). The experimental results of Mitrpant et al show that the survival period of heavy SMA mice treated with ASO is prolonged to 37-126 days, which is significantly longer than that of the control group by 15 days (Mitrpant C, et al, PLoS one, 2013; 8(4): e 62114.18). Another mouse experiment performed by Rigo et al showed that the level of full-length SMN2 gene in the lumbar spinal cord was elevated 3-fold after 7-day continuous infusion or a single lateral ventriculo injection of MOE-ASO into the lateral ventricles of SMA mice and was maintained for about 52 and 36 weeks, respectively (Rigo F, et al, J Pharmacol Exp ther. 2014; 350(1): 46-55.). In addition, Rigo et al have also developed a model experiment on non-human primates by injecting varying doses of MOE-ASO intrathecally into adult macaques to study drug distribution in primates, which showed extensive distribution of MOE-ASO in spinal cord segments, cerebellum, pons and cerebral cortex, especially with the highest concentration of MOE-ASO in lumbar spinal cord, and provided theoretical basis for studies on drug distribution and metabolism in humans in clinical trials (Rigo F, et al, J Pharmacol Exp Ther 2014; 350(1): 46-55.). Medicine candidate Nusinesiresn of MOE-ASO (original name ISIS-SMN)_RX) The distribution and metabolism of the drug in humans were evaluated by intrathecal injection of Nusinorsen in hundreds of SMAl, 2, 3 patients in phase I, II clinical trials (Chiriboga CA, et al neurology, 2016; 86(10): 890-. Subsequent phase III clinical trials prove that Nusines has remarkable clinical curative effect on SMA children patients. Nusineserssen, marketed under the trade name SPINRZA, was approved by the Food and Drug Administration (FDA) in 2016, 12/23, and was the first new drug for the treatment of SMA in children and adults (https:// www.accessdata.fda.gov /).

(3) Small molecule compounds: there are many kinds of small molecule compounds that can increase the level of SMN protein, and the mechanism of action of up-regulating SMN protein by different classes of small molecule compounds is different (Cherry JJ, et al EMBO Mol Med.2013; 5(7): 1035 1050.). Histone deacetylase inhibitor (HDACI) class of drugs that use valproic acid as a research hotspot may increase the activity of transcription factors by remodeling chromatin, altering the structure of transcription factors, increasing the amount of full-length SMN2 transcripts, and consequently increasing the full-length SMN protein level (Mohseni J, et al, Genet Mol biol 2013; 36(3): 299-307.). After upregulation of full-length SMN2 transcripts was observed in initial cell level studies, although clinical trials reported that increased SMN protein levels and improved limb muscle strength were observed in children with SMA2 (Swoboda KJ, et al PLoS one. 2009; 4(5): e5268. pierers S, et al J neuro therapy 2011; 82(8): 850-852.26-27), however, most clinical test results show that the increasing effect of HDACI on SMN protein level and the improvement of limb Muscle strength of SMA2, children patients with type 3 and adult patients with type SMA4 is not obvious (Merei E, et al Neurouscu disord 2004;14 (2):130-135, Darbar IA, et al BMC neurology 2011 11: 36 Kissel JT, et al Muscle nerve 2014; 49(2): 187-192.28-30), so the clinical efficacy and the application prospect of HDACI drugs cannot be fully determined. Small molecule compounds which are screened by a high-throughput screening technology (HTS) and have the effect of increasing the expression of SMN2 gene, such as RNA decapping enzyme inhibitor RG3039, have the function of stabilizing the mRNA structure of SMN2 gene by inhibiting the hydrolysis of m7GPPPN cap structure at the 5' end of mRNA, thereby improving the total amount of SMN protein, improving the motor function of heavy delta 7SMA mice and prolonging the survival period (Lutz CM, et al J Clin invest. 2011; 121(8): 3029-3041. Gogliotti RG, et al Hum Mol Genet. 2013; 22(20): 4084-4101.31-32). At present, the phase I clinical test of RG3039 is finished, and the result shows that the medicine is safe and well tolerated, but the phase II clinical test is stopped, and the pharmaceutical company does not publish specific reasons. Similarly, small molecules R006885247 and LMl070 can also enhance the expression of SMN2 gene, and phase I and phase II clinical trials (Yanglan, et al. Zhonghua J. pediatrics.2016; 54 (8): 634. 637.) have been conducted. Small molecule RO7034067 (RG 7916), which is also an activator of SMN2 gene expression, has been tested for clinical safety assessment in the Netherlands and Japan, and is now ready for clinical safety assessment in the United states (https:// www.clinicaltrials.gov). The SMN2 gene splicing regulators SMN-C1, SMN-C2 and SMN-C3 screened by the improved second-generation HTS technology can promote the expression of the full-length SMN2 gene, and the compounds can pass through a blood brain barrier after being taken orally and have obvious effect in preclinical research, so that the compounds become a new research hotspot. Naryshkin et al found that after SMN-C2 and SMN-C3 were orally administered continuously for 10 days, the SMN protein levels in the brain and the quadriceps femoris of mice were significantly increased, and the weight of heavy Δ 7SMA mice increased close to that of normal mice, and the survival time was significantly prolonged compared with that of untreated mice (Naryshkin NA, et al, science 2014; 345(6197): 688-693.). However, currently, SMN-C1, SMN-C2 and SMN-C3 are in preclinical research. 2. SMN-independent treatment: before irreversible degeneration damage of anterior horn motor neurons of spinal cord, some neuromuscular protection measures are also very important for the maintenance of motor functions of SMA children. Stem cell therapy, nerve cell protection factors, muscle enhancement drugs and other therapeutic methods which do not depend on the improvement of SMN protein expression protect the functions of motor neurons and nerve pathways thereof through different ways, and have certain auxiliary effects on the therapeutic measures for improving the SMN protein.

The stem cell therapy is induced pluripotent stem cell (induced)pluripotentstem cells, iPSCs) are transplanted into the spinal cord after differentiation into motor neurons, replacing motor neurons that have degenerated and degenerated, thereby restoring the function of the neuromuscular system, and clinical studies have shown that this method can improve the motor function of SMAl-type infants (Zanetta C, et al. J Cell Mol Med 2014; 18(2):187-196. Villanova M, et al. Am J Phys Med rehabil 2015; 94 (5): 410-415.34-35) is considered a potential treatment strategy. The neuroprotective factor medicines riluzole and orlistat (Olesoxime) are both used for treating amyotrophic lateral sclerosis, and the results of SMA clinical tests show that riluzole can prolong the survival time of SMA1 children, but has no obvious effect on improving the motor function (Russman BS, et al. Arch neurol. 2003; 60(11): 1601 and 1603), and the orlistat can better maintain the motor function of SMA2 and 3 children, so the United states and European Union agree to use the orlistat for the auxiliary treatment of SMA. Another drug for multiple sclerosis treatment, 4-aminopyridine (4-AP, Ampyra), blocks potassium channels to maintain motor neuron excitability and thus increase muscle contraction (Bordet T, et al J Pharmacol Exp ther. 2007; 322(2): 709-. In addition, the skeletal troponin activator CK-2127107 (Hwee DT, et al J Pharmacol Exp ther. 2015; 353(1): 159. 168.) used for the study of heart failure mediated skeletal myopathy in recent years has been subjected to the study of SMA treatment for improving skeletal muscle strength and improving muscular atrophy and weakness of SMA children, and CK-2127107 has been subjected to the clinical trial of SMA phase II. Since the localization of the causative gene of SMA in 1995, the pathogenesis of this disease has been a hot spot explored by scientists. Although effective treatments are still lacking, the fruitful results obtained from therapeutic studies based on the increased expression of SMN proteins hold promise for the treatment of SMA. The gene therapy scAAV9-SMN has the advantages of meeting the treatment requirement through single large-dose administration, avoiding multiple injections, and showing obvious effect and good safety of the method by animal experiments, but the small virus dosage (up to 2 multiplied by 10) in clinical tests¹⁴vg/kg) may present safety issues, and still deserves further attention. And how to produce a large amount of virus vectors to meet the treatment requirement of SMA children patients in the world is also a difficult problem to be solved urgently. SPINRZA can specifically act on SMN2 gene, so the side effect is less, the effect is obvious, but the SPINRZA can not effectively pass through blood brain barrier, repeated intrathecal injection is needed, and the compliance of the children patients is reduced. The small molecular compounds have the advantages of convenient administration, act on the central nervous system through the blood brain barrier after oral administration, and have the defect that the adverse reaction to other genes cannot be determined because various small molecular compounds do not specifically act on SMN genes. In addition, animal experiments and clinical trials suggest that SMA treatment has a relatively narrow time window, and how to determine the optimal time for administration is also a key issue. Therefore, a new SMA treatment medicine needs to be developed, and a new choice is provided for vast SMA patients.

In the invention, a gene therapy strategy is adopted, and a series of new SMA gene therapy candidate medicines are designed aiming at the possible limitations of SMA gene medicines. Firstly, a novel SMN1 gene expression frame structure is designed, a plurality of promoter elements with short sequences and high expression level are independently designed, and the sequence of the SMN1 gene coding region is optimized, so that the expression level of the SMN1 gene after being transferred into a human body is improved. Secondly, we also tried to add a human miR-122 target sequence into the 3 'UTR (3' -untranslated region) of the SMN1 gene expression cassette, and utilize the characteristic of high expression of miR-122 in normal liver (joint C. RNA biol. 2012; 9(2): 137-. Then we also provide an autonomously designed double-stranded AAV vector structure, which is mainly characterized in that deletion mutation is carried out on one or two Inverted Terminal Repeat (ITR) in the AAV vector, thus being helpful for improving the in vivo transduction efficiency of the AAV vector and improving the expression level of the SMN1 gene introduced into the body (patent application No. CN201510931560.3, Zhou Q, et al Sci Rep.2017; 7(1): 5432.). Again we also provided other serotype AAV drug designs than AAV9, specifically AAV5 and AAVrh10, both of which can efficiently deliver their carried SMN1 expression cassette to the nervous system. Finally, we compared the effects of different modes of administration (e.g., intravenous, intrathecal, and intracerebroventricular) on the safety and efficacy of the designed drugs.

Adeno-associated virus (AAV) is known as found in adenovirus preparations (atcheson RW,et al. Science. 1965; 149: 754-756.Hoggan MD, et alproc Natl Sci USA 1966; 55: 1467-. AAV is a member of the family parvoviridae (subvirus), and comprises multiple serotypes, the genome of which is single-stranded DNA (Rose JA,et alproc Natl Acad Sci USA 1969; 64: 863-. AAV is a dependent virus, requiring other viruses such as adenovirus, herpes simplex virus, and human papilloma virus (Geoffroy MC,et alcurr Gene ther 2005, (5 (3): 265-271), or an auxiliary factor provides an auxiliary function to copy. In the absence of helper virus, AAV infects cells and its genome integrates into the cell chromosome to become latent (Chiorini JA,et alcurr Top Microbiol Immunol. 1996; 218:25-33.) without production of progeny virus.

The first AAV virus isolated was serotype 2 AAV (AAV 2) (atcheson RW,et alscience 1965, 149: 754-. The AAV2 genome is about 4.7kb long, with Inverted Terminal Repeats (ITRs) of length 145bp at both ends of the genome, in a palindromic-hairpin structure (Lusby E,et alj Virol, 1980; 34: 402-409). There are two large Open Reading Frames (ORFs) in the genome, encoding the rep and cap genes, respectively. The full-length genome of AAV2 has been cloned into an e.coli plasmid (Samulski RJ,et al. Proc Natl Acad Sci USA. 1982; 79: 2077-2081. Laughlin CA, et al. Gene. 1983; 23: 65-73.)。

ITRs are cis-acting elements of the AAV vector genome that play important roles in integration, rescue, replication, and genome packaging of AAV viruses (Xiao X,et alj Virol, 1997, (71) (2) 941-948). The ITR sequences contain a Rep protein binding site (RBS) and a terminal melting site, trs (terminal resolution site), which are recognized by Rep protein binding and nicked at trs (Linden RM,et alproc Natl Acad Sci USA 1996; 93(15): 7966-. ITR sequences may also form unique "T" alphabetical secondary structures that play an important role in the life cycle of AAV viruses (Ashktorab H,et al. J Virol. 1989; 63(7): 3034-3039.)。

the remainder of the AAV2 genome can be divided into 2 functional regions, the rep and cap gene regions (Srivastava a,et alj Virol, 1983, 45(2), 555-. The Rep gene region encodes four Rep proteins, Rep78, Rep68, Rep52 and Rep 40. Rep proteins play an important role in replication, integration, rescue and packaging of AAV viruses. Wherein Rep78 and Rep68 specifically bind to terminal melting sites trs (terminal resolution site) and the GAGY repeat motif in ITRs (Huser D,et alPLoS Patholog.2010, 6(7) e1000985. the replication process of AAV genome from single strand to double strand is initiated. The trs and GAGC repeat motifs in the ITRs are central to replication of the AAV genome, and therefore although the ITR sequences are not identical in all serotypes of AAV virus, both hairpin structures are formed and Rep binding sites are present. The AAV2 genome map has p19 promoter at position 19, and expresses Rep52 and Rep40, respectively. Rep52 and Rep40 have no function of binding to DNA, but have ATP-dependent DNA helicase activity. The cap gene encodes the capsid proteins VP1, VP2, and VP3 of AAV virus. Of these, VP3 has the lowest molecular weight but the highest number, and the ratio of VP1, VP2, and VP3 in mature AAV particles is approximately 1:1: 10. VP1 is essential for the formation of infectious AAV; VP2 assists VP3 in entering the nucleus; VP3 is the major protein that makes up AAV particles.

With the understanding of the life cycle of AAV and its related molecular biological mechanism, AAV is transformed into one efficient foreign gene transferring tool, AAV vector. The modified AAV vector genome only contains the ITR sequence of AAV virus and the exogenous gene expression frame carrying transport, and Rep and Cap proteins required by virus package are provided in trans by exogenous plasmidAnd the possible harm caused by packaging rep and cap genes into the AAV vector is reduced. Moreover, the AAV virus itself is not pathogenic, making the AAV vector one of the most recognized safe viral vectors. Deletion of the D sequence and the trs (tertiary resolution site) sequence in the ITR sequence on one side of the AAV enables self-complementation of the genome carried by the packaged recombinant AAV vector to form double chains, thus remarkably improving the in vitro and in vivo transduction efficiency of the AAV vector (Wang Z,et al. Gene Ther. 2003;10(26):2105-2111. McCarty DM, et algene ther 2003, 10(26) 2112-2118). The resulting packaged virus becomes a scAAV (self-complementary AAV) virus, a so-called double-stranded AAV virus. Unlike ssAAV (single-stranded AAV), a classical AAV virus, in which neither ITR is mutated at both sides. The packaging capacity of scAAV virus is smaller, only half of the packaging capacity of ssAAV, about 2.2kb-2.5kb, but transduction efficiency is higher after infecting cells. AAV viruses are numerous in serotype, different serotypes having different tissue infection tropism, and thus the use of AAV vectors enables the transport of foreign genes to specific organs and tissues (Wu Z,et almol ther 2006, 14(3) 316-. Some serotype AAV vectors can also cross the blood brain barrier, leading foreign genes into brain neurons, providing the possibility for gene transduction targeting the brain (Samaranch L,et alhum Gene ther, 2012, 23(4) 382. 389.). In addition, AAV vectors have stable physicochemical properties, and exhibit strong tolerance to acids and bases and high temperatures (Gruntman AM,et alhum Gene their methods 2015, 26(2): 71-76), it is easy to develop biological products with higher stability.

AAV vectors also have relatively mature packaging systems, facilitating large-scale production. At present, the AAV vector packaging system commonly used at home and abroad mainly comprises a three-plasmid cotransfection system, a packaging system taking adenovirus as a helper virus, a packaging system taking Herpes simplex virus type 1 (HSV 1) as a helper virus and a packaging system based on baculovirus. Among them, the three plasmid transfection packaging system is the most widely used AAV vector packaging system because of no need of auxiliary virus and high safety, and is also the mainstream production system in the world at present. Slightly deficient, is the absence of an efficient large-scale transfection methodThe application of the three-plasmid transfection system in the large-scale preparation of AAV vectors is limited. Yuan et al established an AAV large-scale packaging system with adenovirus as the helper virus (Yuan Z,et alhum Gene Ther, 2011,22(5): 613-. HSV1 is another type of AAV vector packaging system that has been used more widely as a packaging system for helper viruses. Almost simultaneously, Wushijia and Conway et al internationally proposed the packaging strategy of AAV2 vector with HSV1 as helper virus (Wushijia, Wu soldier et al scientific bulletin, 1999, 44 (5): 506-509. Conway JE,et algene Ther, 1999,6: 986-. Subsequently, Wustner et al proposed an AAV5 vector packaging strategy with HSV1 as a helper virus (Wustner JT,et almol Ther, 2002,6(4): 510-. On the basis, Booth et al utilize two HSV1 to respectively carry the rep/cap gene of AAV and Inverted terminal sequence (ITR)/exogenous gene expression cassette of AAV, then two recombinant HSV1 viruses are co-infected with production cell, packaged to produce AAV virus (Booth MJ,et algene Ther,2004,11: 829-. Thomas et al further established the suspension cell system for AAV production of bis HSV1 virus (Thomas DL,et algene Ther,2009,20: 861-870), enabling larger scale AAV virus production. In addition, Urabe and the like construct a baculovirus packaging system of AAV vectors by using three baculoviruses to respectively carry AAV structural, non-structural and ITR/exogenous gene expression cassettes. Considering the instability of baculovirus carrying foreign genes, the number of baculovirus required in the production system is subsequently reduced, gradually from the first requiring three baculovirus to the second requiring two or one baculovirus (Chen H. Mol ther.2008;16(5):924-,et alj Invertebr Pathol, 2011,107 Suppl: S80-93.) and a baculovirus plus one strain inducing cell line strategy (Mietzsch M,et al. Hum Gene Ther. 2014;25:212-222. Mietzsch M, et alhum Gene ther 2015, 26(10) 688 697. Each packaging system has various characteristics, and can be selected as required.

Due to the above characteristics, AAV vectors have been developedBecomes an exogenous gene transfer tool which is widely applied to gene therapy, in particular to gene therapy of genetic diseases. By 11 months 2017, there are 204 approved gene therapy clinical trials of AAV vectors worldwide (http:// www.abedia.com/willey/vectors. More importantly, the AAV vector-based lipoprotein lipase gene therapy drug Glybera was approved by the european drug administration in 2012 to be marketed as the first gene therapy drug approved in the western world (Yl ä -herttaala S).Mol Ther2012, 20(10) 1831 and 1832); the American FDA approved congenital amaurosis (caused by RPE65 gene mutation) gene therapy medicament Luxturna is marketed in 19.12.2017, and becomes the first gene therapy medicament for the rare diseases in the United states (https:// www.fda.gov/news events/news room/presentation/ucm 589467. htm). Hemophilia B (Kay MA, et al.Nat GenetThe AAV vector gene therapy medicaments of 2000, 24(3), 257 and 261) all have good clinical test effects, are expected to be sold in the near future and benefit a large number of patients.

In the invention, the AAV vector is selected to carry the SMN1 gene expression cassette, and is mainly based on the following characteristics of the AAV vector. For one, AAV vectors retain only the two ITR sequences required for packaging in wild-type virus, and do not contain the protein-encoding genes in the wild-type virus genome (salenik M,et almicrobiol spectra. 2015; 3(4), which is low in immunogenicity. Secondly, AAV achieves sustained stable expression of the gene-carrying reading frame, usually in the form of non-integrated extrachromosomal genetic material (Chen ZY,et almol ther 2001, 3(3) 403-. Third, AAV vectors transduce the central nervous system efficiently by intravenous, intrathecal and intraventricular injection (Foust KD, et al Nat Biotechnology. 2009; 27: 59-65. Yang B, et al Mol Ther. 2014; 22(7): 1299-&Psychiatry 2016; 87(Suppl 1): A91.2-A91.Gray SJ, et al Gene ther.2013; 20:450-Bai, restoring the physiological function of the body.

In order to reduce the safety risk possibly brought by the overexpression of SMN in the liver during intravenous administration, the characteristic of specific high expression of miR-122 in normal liver (joining C. RNA biol. 2012; 9(2): 137-. By means of the miRNA gene expression inhibition principle (Kim VN. Nat Rev Mol Cell biol.2005;6(5): 376-385.), the miR-122 molecules in the liver cells can inhibit the expression of SMN protein and obviously reduce the expression of the SMN protein in the liver.

miRNAs (microRNAs) are single-stranded non-coding RNAs of 18 to 25 nucleotides (nt) in length that are widely found in humans and animals (Bartel DP. Cell. 2004; 116: 281-297. Kim VN. Nat Rev Mol Cell biol.2005; 6: 376-385.). miRNA was first found in caenorhabditis elegans (c. elegans) in 1993 (Lee RC,et al. Cell. 1993; 75: 843-854. Wightman B, et alcell. 1993; 75: 855 · 862.). The lin-4 gene in elegans is capable of down-regulating the expression of the lin-14 gene, but the encoded product of the lin-4 gene is not a protein, but is a small RNA molecule, indicating that the small RNA molecule itself encodes for the ability to regulate the expression of the gene. Subsequently, a number of similar small RNA molecules were sequentially found in different species and cells (Lagos-Quintana M,et al. Science. 2001; 294: 853-858. Lau NC, et al. Science. 2001; 294: 858-862. Lee RC, et alscience 2001, 294: 862-864), mirnas began to become a collective term for this class of small RNAs. mirnas regulate the expression of approximately 60% of genes in humans (Lewis BP,et al. Cell. 2005;120: 15-20. Friedman RC, et algenome res. 2009; 19: 92-105), plays an important role in a variety of physiological and pathological processes (carteon M,et al. Cell Cycle. 2007; 6: 2127-2132. Ambros V. Cell. 2003; 113:673-676. Schichel R, et al. Oncogene. 2008; 27: 5959-5974.)。

miRNA genes are typically located in exons, introns, and intergenic regions of the genome (Olena AF,et al. J Cell Physiol. 2010; 222: 540-545. Kim VN, et altrends Genet 2006; 22: 165-173.). Intracellular production of miRNAsThe production process is as follows (Winter J,et alnat Cell biol. 2009;11: 228-. Firstly, in a cell nucleus, miRNA genes are initiated to be transcribed by RNA polymerase II or III to generate an initial product pri-microRNA; the pri-microRNA self-folding partial sequence forms a stem-loop structure. Subsequently, the processing complex consisting of ribonuclease III Drosha and DGCR8 molecules acts on the pri-microRNA, cutting off the excess sequence, leaving around 60nt of stem-loop structure, the precursor miRNA molecule pre-microRNA (Lee Y,et al. Nature. 2003;425: 415-419. Denli AM, et al. Nature. 2004;432: 231-235. Gregory RI, et al. Nature. 2004; 432:235-240. Han J, et al. Genes Dev. 2004; 18: 3016-3027. Landthaler M, et alcurr biol. 2004;14: 2162-. The pre-microRNA then passes from the nucleus into the cytoplasm with the aid of the transporter Exportin-5 (Lund E,et al. Science. 2003; 303: 95-98. Yi R, et al. Genes Dev. 2003;17: 3011-3016. Bohnsack MT, et alRNA, 2004; 10: 185-191.) whose stem-loop structure is processed by Dicer enzyme to remove the loop portion and become a double-stranded RNA molecule (Jiang F,et al. Genes Dev. 2005; 19: 1674-1679. Saito K, et alPLoS biol.2005; 3: e 235.). Finally, the double-stranded RNA molecule is bound by a protein factor such as AGO2, one strand of which is degraded, and the other strand of which forms an RNA-induced silencing complex (RISC) with the protein factor. RISC recognizes target sequences in mRNA, reduces mRNA expression levels by degrading mRNA molecules, promoting 3' end de-adenylation of mRNA molecules, and inhibiting translation, regulates gene expression at post-transcriptional levels (Storz G,et al. Curr Opin Microbiol.2004; 7: 140-144. Fabian MR, et al. Annu Rev Biochem. 2010; 79: 351-379. Valencia-Sanchez MA, et algenes Dev 2006; 20: 515-. Therefore, the expression of a foreign gene in a cell to be introduced can be effectively suppressed by inserting a target sequence of a miRNA into the 3' UTR (untranslated region) of the foreign gene using the miRNA highly expressed in the cell.

According to the design thought, a plurality of recombinant AAV viruses carrying SMN1 gene expression frames are prepared, and rAAV-Fluc control viruses carrying firefly luciferase gene (Fluc) coding frames are designed and prepared. These viruses were injected into SMA mouse models at equal doses by intravenous, intrathecal, or intracerebroventricular injection, and the like, to evaluate the effectiveness of designing recombinant AAV viruses carrying SMN1 gene expression cassettes. The result shows that compared with rAAV-Fluc control virus, the recombinant AAV carrying the SMN1 gene expression frame can efficiently transduce the central nervous system after being injected by intravenous injection, intrathecal injection or intracerebroventricular injection, and the like, and the SMN protein is expressed and generated in neurons, thereby recovering the physiological function of the neurons and obviously prolonging the survival period of a mouse model. Compared with the existing AAV vector-based SMA gene therapy strategy, especially the AVXS-101 which is already in clinical trial, the unique SMN1 expression cassette design in the invention improves the expression level of the SMN1 expression cassette in vivo and reduces the dosage of virus. In combination with the mutant ITR structure invented by us, the expression cassette of SMN1 gene is inserted into AAV vector containing mutant ITR structure, so that the expression efficiency in preparing virus transductor is further improved. In the invention, besides AAV9, the possibility of AAV5 and AAVrh10 for SMA gene therapy is evaluated, and the result shows that AAV5 and AAVrh10 can efficiently transfer the central nervous system and express SMN protein in the same application prospect as AAV9 after being injected into the body in intravenous injection, intrathecal injection or intracerebroventricular injection mode like AAV 9. Although the transduction efficiency of AAV5 is slightly lower than that of AAV9 and AAVrh10, the same transduction efficiency as AAV9 and AAVrh10 can be achieved by increasing the dose. In the invention, a plurality of possible administration modes are explored, and intrathecal and intracerebroventricular injection modes can realize high-efficiency transduction of neurons under a lower administration dosage, express to generate SMN protein, effectively relieve the nervous system injury effect mainly caused by SMN1 gene mutation, and provide a new administration mode selection for SMA gene therapy. Intravenous injection of AAV viral vectors results in efficient expression of foreign genes in the liver, which may carry safety risks. We also design an SMN1 expression box containing a human miR-122 target sequence, prepare a recombinant AAV vector containing the expression box, and after intravenous injection, the SMN protein expression level in the liver is obviously reduced, while the SMN protein expression level in the nervous system is not affected, so that a new choice is provided for reducing the safety risk possibly brought by the SMN protein overexpression in the liver.

In summary, we designed and produced a variety of recombinant AAV vectors containing the SMN1 expression cassette in the present invention, and in vivo evaluation results indicated that these structural designs could achieve the desired therapeutic effect at lower doses than reported in the literature. We also evaluate the influence of different administration modes on the treatment effect, and find that the intrathecal, intracerebroventricular and intravenous injection modes in animal models can achieve better treatment effect. We also enhanced the safety of the designed drug when injected intravenously by introducing the human miR-122 target sequence. We also obtained two new AAV vector serotypes, AAV5 and AAVrh10, that could be used for SMA gene therapy. These all provide new options for gene therapy of SMA.

Disclosure of Invention

In view of the above, the present invention provides a series of recombinant AAV viral vectors carrying artificially designed SMN1 gene expression cassettes and their use in SMA therapy. The artificially designed SMN1 gene expression cassette can be put into the body to efficiently express and generate the SMN1 protein, and the dosage of AAV which achieves the efficacy of SMA treatment is reduced. Through selection of recombinant AAV virus vector serotypes, mutation transformation of main cis-acting element inverted terminal repetitive sequences and selection of proper AAV virus vectors, the purpose of artificially designing SMN1 gene expression cassettes to be efficiently transported to target organs and tissues is achieved. The SMA model mouse is transduced by the recombinant AAV vector containing the artificially designed SMN1 gene expression frame, so that the survival period of the SMA mouse can be prolonged remarkably, the body weight of the SMA mouse can be increased, and the development potential of SMA therapeutic drugs can be displayed.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an artificially designed SMN1 gene expression cassette which is characterized by comprising sequence elements such as an artificially designed promoter, a codon optimized SMN1 coding region sequence, a human miR-122 target sequence and the like and related combination forms thereof. Specifically, in example 1 and example 3, 5 promoters were designed artificially, the differences in transduction efficiency of the 5 promoters in vitro and in vivo were evaluated using a firefly luciferase (Fluc) reporter gene, and 2 promoters useful for the expression design of SMN1 gene were screened according to in vitro and in vivo experimental results. In example 4, the influence of codon optimization of the coding region sequence of SMN1 on the effectiveness of the SMN1 expression cassette on SMA model animals is compared, and the optimized SMN1 coding region sequence is found to show better effectiveness. In example 4, the influence of the human miR-122 target sequence introduced into the SMN1 gene expression cassette on the action effect of the SMN1 gene expression cassette is also evaluated, and the result shows that the human miR-122 target sequence inserted into the expression cassette can obviously reduce the expression of SMN1 protein in the liver and reduce the hepatotoxicity possibly brought by the over-expression of SMN 1.

The invention provides a recombinant AAV carrying an artificially designed SMN1 gene expression cassette, which is characterized in that a reverse terminal repeat sequence in the recombinant AAV is subjected to mutation such as deletion of a B-B 'sequence, a C-C' sequence, a D sequence and the like, so that the prepared recombinant AAV has higher transduction efficiency and can express an SMN1 protein more rapidly after being transduced into a body. Specifically, in example 1, we constructed a variety of AAV plasmid vectors containing mutated inverted terminal repeats. In example 3, we compared the in vivo expression levels of the AAV viruses constructed in example 1 and evaluated whether the mutants helped to increase the therapeutic effect of AAV viruses containing the SMN1 gene expression cassette on SMA model mice. The result shows that the deletion of the B-B 'sequence, the C-C' sequence and the D sequence in the AAV inverted terminal repeat sequence is helpful for improving the in vivo transduction efficiency of the recombinant AAV prepared by packaging and enhancing the effectiveness of SMA gene drug design.

The recombinant AAV carrying the artificially designed SMN1 gene expression frame provided by the invention is also characterized in that the recombinant AAV comprises a plurality of serotypes, such as AAV5, AAV9, AAVrh10 and the like, different serotypes of AAV can penetrate through the blood brain barrier, the SMN1 gene expression frame is transported to neurons, and SMN1 protein is generated by expression, so that the effect of treating SMA diseases is achieved. Specifically, in example 6, the effect of different serotypes of AAV viral vectors carrying SMN1 gene expression cassettes on the effectiveness of SMA model mice is compared, and the result shows that the survival period of the model can be prolonged and the weight of the model can be increased despite the difference between different serotypes, so that the potential for treating SMA diseases is shown.

The SMA gene therapy medicine provided by the invention is characterized in that the medicine is a recombinant AAV carrying an artificially designed SMN1 box. The virus can be used for expressing and generating human SMN1 protein in an SMA model mouse for a long time continuously after once administration, so that the physiological function of the model mouse is recovered, and the life cycle is prolonged.

The SMA gene therapy medicine provided by the invention is also characterized in that the administration mode of the medicine can be intravenous injection, intrathecal injection or intracerebroventricular injection. Different injection modes can affect the dosage of the medicine, but can prolong the survival time of SMA model animals.

The important original experimental materials used in the present invention are as follows:

pHelper plasmid, derived from AAV Helper Free System (Agilent Technologies, USA), was purchased from Agilent Technologies, Inc. and stored. The plasmid contains three plasmids to co-transfect HEK293 cells to prepare adenovirus-derived helper function genes E2A, E4, VA RNA and the like required by recombinant AAV.

pAAV-RC plasmid, derived from AAV Helper Free System (Agilent Technologies, USA), was purchased from Agilent Technologies, Inc. and stored. The pAAV-RC plasmid contains the Rep and cap genes of AAV2 intact, and provides the 4 Rep proteins (Rep 78, Rep68, Rep52 and Rep 40) and AAV2 coat proteins necessary for packaging in the preparation of recombinant AAV2 virus by three-plasmid co-transfection packaging.

The pAAV-R2C5 plasmid was constructed and stored by this company. The plasmid sequence pAAV-R2C5 is obtained by using pAAV-RC plasmid in AAV Helper Free systems (Agilent Technologies, USA) as basic skeleton and replacing sequences 2013 to 4220 in pAAV-RC plasmid with coat protein coding sequence Cap5 (sequences 2207 to 4381 in genome) in AAV genome (GenBank ID: NC-006152.1). The simple construction process is that pAAV-R2C5 plasmid sequence information is obtained according to the above thought, sequences between HindIII and PmeI restriction sites in the pAAV-R2C5 plasmid are artificially synthesized, and the sequences between HindIII and PmeI of the pAAV-RC plasmid are replaced by the synthetic sequences by adopting a standard molecular cloning method to obtain the pAAV-R2C5 plasmid. The pAAV-R2C5 plasmid contains the cap gene of AAV5 and the Rep gene of AAV2 completely, and 4 Rep proteins (Rep 78, Rep68, Rep52 and Rep 40) and AAV5 coat proteins which are necessary for virus packaging are provided in the preparation of recombinant AAV5 virus by three-plasmid co-transfection and packaging.

The pAAV-R2C9 plasmid was constructed and stored by this company. The pAAV-RC plasmid in AAV Helper Free System (Agilent Technologies, USA) is used as basic skeleton, and the sequences 2013 to 4220 in pAAV-RC plasmid are replaced by AAV9 coat protein coding sequence (GenBank ID: AY 530579), so that pAAV-R2C9 plasmid is obtained. The simple construction process is that pAAV-R2C9 plasmid sequence information is obtained according to the thought, a sequence between HindIII and PmeI restriction sites in the pAAV-R2C9 plasmid is artificially synthesized, and a standard molecular cloning method is adopted to replace the sequence between the HindIII and PmeI restriction sites of the pAAV-RC plasmid by the synthetic sequence to obtain the pAAV-R2C9 plasmid. The pAAV-R2C9 plasmid contains the cap gene of AAV9 and the Rep gene of AAV2 in a complete form, and 4 Rep proteins (Rep 78, Rep68, Rep52 and Rep 40) and AAV9 coat proteins which are necessary for packaging are provided in the preparation of recombinant AAV9 virus by three-plasmid co-transfection packaging.

The pAAV-R2C10 plasmid was constructed and stored by this company. The sequence from 2013 to 4220 in pAAV-RC plasmid was replaced by the coding sequence of AAVrh10 coat protein (GenBank ID: AY 243015.1) using pAAV-RC plasmid in AAV Helper Free System (Agilent Technologies, USA) as basic skeleton, and pAAV-R2C10 plasmid was obtained. The simple construction process is that pAAV-R2C10 plasmid sequence information is obtained according to the thought, a sequence between HindIII and PmeI restriction sites in the pAAV-R2C10 plasmid is artificially synthesized, and a standard molecular cloning method is adopted to replace the sequence between the HindIII and PmeI restriction sites of the pAAV-RC plasmid by the synthetic sequence to obtain the pAAV-R2C10 plasmid. The pAAV-R2C10 plasmid contains the cap gene of AAVrh10 and the Rep gene of AAV2 completely, and provides 4 Rep proteins (Rep 78, Rep68, Rep52 and Rep 40) and AAVrh10 coat protein which are necessary for packaging in the preparation of recombinant AAVrh10 virus through three-plasmid co-transfection and packaging.

The pAAV-DJ plasmid, comprising the cap gene of the intact AAVDJ and the Rep gene of AAV2, provides the 4 Rep proteins (Rep 78, Rep68, Rep52 and Rep 40) and the AAVDJ coat protein necessary for packaging in the preparation of recombinant AAVDJ viruses by three-plasmid co-transfection packaging. Purchased from Cell Biolabs, usa and stored.

Control mice, C57BL/6J mice, purchased from Beijing Huafukang Biotech GmbH, used as wild-type controls for animal experiments.

The GM03813 Cell line, purchased from Coriell Cell reproduction, USA, was derived from a Cell line of SMA type I patients (Coovert DD, et al. Hum Mol Genet. 1997; 6: 1205-1214.). The invention is used as an in vitro experimental cell strain for screening a promoter.

SMN2^+/+, SMNΔ7^+/+, smn^+/-Mice, purchased from Jackson laboratory, USA, and numbered 005025, 5 males and females, were bred to obtain SMN2^+/+, SMNΔ7^+/+, smn^-/-Mouse, used as animal evaluation model for the design of SMA gene medicine. Propagation and genotyping of the models is described in reference to the Jackson laboratory. SMN2^+/+, SMNΔ7^+/+, smn^-/-The mice are referred to in the examples as "SMA model mice" or "SMA mice".

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a schematic diagram of the pAAV2neo vector structure. The AAV vector pAAV2neo (Dong X, et al, PLoS ONE. 2010; 5(10): e 13479.) with both ITRs on both sides being 145bp wild-type ITRs was stored by this company. ITR, inverted terminal repeat, length 145 bp. CMV promoter, human cytomegalovirus early promoter. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame. XhoI, KpnI, EcoRI, SalI, BglII, BamHI and ApaI are all restriction sites.

FIG. 2 is a schematic diagram of the structure of pAAV2neo-Fluc vector. ITR, inverted terminal repeat, length 145 bp. CMV promoter, human cytomegalovirus early promoter. Fluc, firefly luciferase gene coding sequence. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame.

FIG. 3 is a schematic diagram of the structure of pAAV2neo-CAS-Fluc vector. ITR, inverted terminal repeat, length 145 bp. CAS promoter, an artificially designed synthetic promoter, the sequence information of which is detailed in SEQ ID No. 1. Fluc, firefly luciferase gene coding sequence. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame.

FIG. 4 is a schematic diagram of the vector structure of pAAV2 neo-CAT-Fluc. ITR, inverted terminal repeat, length 145 bp. CAT promoter, artificially synthesized promoter, and the sequence information is detailed in SEQ ID No. 2. Fluc, firefly luciferase gene coding sequence. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame.

FIG. 5 schematic diagram of pAAV2neo-CAR-Fluc vector structure. ITR, inverted terminal repeat, length 145 bp. CAR promoter, artificially designed and synthesized promoter, and the sequence information is detailed in SEQ ID No. 3. Fluc, firefly luciferase gene coding sequence. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame.

FIG. 6 is a schematic diagram of the structure of pAAV2neo-CAP-Fluc vector. ITR, inverted terminal repeat, length 145 bp. CAP promoter, an artificially designed and synthesized promoter, and the sequence information is detailed in SEQ ID No. 4. Fluc, firefly luciferase gene coding sequence. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame.

FIG. 7 is a schematic diagram of the structure of pAAV2neo-CA-Fluc vector. ITR, inverted terminal repeat, length 145 bp. CA promoter, human giant cell enhancer and chicken beta-actin promoter, and the sequence information is shown in SEQ ID No. 5. Fluc, firefly luciferase gene coding sequence. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame.

FIG. 8 schematic diagram of the structure of the pscAAV-CA-Fluc vector. ITR, inverted terminal repeat, length 145 bp. Δ ITR, deletion of the inverted terminal repeat of the D sequence, sequence information detailed in SEQ ID No. 6. CA promoter, human cytomegalovirus gene enhancer and chicken beta-actin promoter, and the sequence information is shown in SEQ ID No. 5. Fluc, firefly luciferase gene coding sequence. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame.

FIG. 9 schematic of the pscAAV-CAR-Fluc vector structure. ITR, inverted terminal repeat, length 145 bp. Δ ITR, deletion of the inverted terminal repeat of the D sequence, sequence information detailed in SEQ ID No. 6. CAR promoter, a human designed promoter, and the sequence information is detailed in SEQ ID No. 3. Fluc, firefly luciferase gene coding sequence. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame.

FIG. 10 schematic diagram of the pscAAV-U-CAR-Fluc vector structure. U-ITR, and inverted terminal repetitive sequences of B-B 'and C-C' sequences are deleted, and the sequence information is detailed in SEQ ID No. 8. Delta U-ITR, U-ITR sequence of deletion D sequence, and the sequence information is detailed in SEQ ID No. 10. CAR promoter, a human designed promoter, and the sequence information is detailed in SEQ ID No. 3. Fluc, firefly luciferase gene coding sequence. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame.

FIG. 11 schematic diagram of the structure of the pscAAV-CA-SMN1 vector. ITR, inverted terminal repeat, length 145 bp. Δ ITR, deletion of the inverted terminal repeat of the D sequence, sequence information detailed in SEQ ID No. 6. CA promoter, human cytomegalovirus gene enhancer and chicken beta-actin promoter, and the sequence information is shown in SEQ ID No. 5. SMN1, human motor neuron survival gene 1 coding sequence, and the sequence information is detailed in SEQ ID No. 12. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame.

FIG. 12 schematic diagram of the vector structure of pscAAV-CA-cosMN 1. ITR, inverted terminal repeat, length 145 bp. Δ ITR, deletion of the inverted terminal repeat of the D sequence, sequence information detailed in SEQ ID No. 6. CA promoter, human giant cell enhancer and chicken beta-actin promoter, and the sequence information is shown in SEQ ID No. 5. coSMN1, human expression optimized human motoneuron survival gene 1 coding sequence, sequence information is detailed in SEQ ID No. 13. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame.

FIG. 13 schematic representation of the pscAAV-CAR-SMN1 vector structure. ITR, inverted terminal repeat, length 145 bp. Δ ITR, deletion of the inverted terminal repeat of the D sequence, sequence information detailed in SEQ ID No. 6. CAR promoter, a promoter sequence designed by people, and the sequence information is detailed in SEQ ID No. 3. SMN1, human motor neuron survival gene 1 coding sequence, and the sequence information is detailed in SEQ ID No. 12. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame.

FIG. 14 schematic of the pscAAV-CAR-cosMN1 vector structure. ITR, inverted terminal repeat, length 145 bp. Δ ITR, deletion of the inverted terminal repeat of the D sequence, sequence information detailed in SEQ ID No. 6. CAR promoter, a promoter sequence designed by people, and the sequence information is detailed in SEQ ID No. 3. coSMN1, human expression optimized human motoneuron survival gene 1 coding sequence, sequence information is detailed in SEQ ID No. 13. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame.

FIG. 15 schematic of the pscAAV-U-CAR-cosMN1 vector structure. U-ITR, and inverted terminal repetitive sequences of B-B 'and C-C' sequences are deleted, and the sequence information is detailed in SEQ ID No. 8. Delta U-ITR, U-ITR sequence of deletion D sequence, and the sequence information is detailed in SEQ ID No. 10. CAR promoter, a human designed promoter, and the sequence information is detailed in SEQ ID No. 3. coSMN1, human expression optimized human motoneuron survival gene 1 coding sequence, sequence information is detailed in SEQ ID No. 13. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame.

FIG. 16 schematic diagram of the pscAAV-CAR-SMN1-122T vector structure. ITR, inverted terminal repeat, length 145 bp. Δ ITR, deletion of the inverted terminal repeat of the D sequence, sequence information detailed in SEQ ID No. 6. CAR promoter, a promoter sequence designed by people, and the sequence information is detailed in SEQ ID No. 3. SMN1, human motor neuron survival gene 1 coding sequence, and the sequence information is detailed in SEQ ID No. 12. miR-122T, a target sequence completely complementary to human miR-122. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame.

FIG. 17 schematic of the pscAAV-CAR-cosMN1-122T vector structure. ITR, inverted terminal repeat, length 145 bp. Δ ITR, deletion of the inverted terminal repeat of the D sequence, sequence information detailed in SEQ ID No. 6. CAR promoter, a promoter sequence designed by people, and the sequence information is detailed in SEQ ID No. 3. coSMN1, human expression optimized human motoneuron survival gene 1 coding sequence, sequence information is detailed in SEQ ID No. 13. miR-122T, a target sequence completely complementary to human miR-122. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame.

FIG. 18 schematic diagram of the pscAAV-U-CAR-cosMN1-122T vector structure. U-ITR, and inverted terminal repetitive sequences of B-B 'and C-C' sequences are deleted, and the sequence information is detailed in SEQ ID No. 8. Delta U-ITR, U-ITR sequence of deletion D sequence, and the sequence information is detailed in SEQ ID No. 10. CAR promoter, a human designed promoter, and the sequence information is detailed in SEQ ID No. 3. coSMN1, human expression optimized human motoneuron survival gene 1 coding sequence, sequence information is detailed in SEQ ID No. 13. miR-122T, a target sequence completely complementary to human miR-122. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame.

FIG. 19 shows the comparison of the expression efficiency of different promoters in HEK293 cells. The expression efficiency of different promoters is compared by taking the firefly luciferase Fluc as a reporter gene. Packaging to obtain the AAVDJ virus carrying Fluc expression frames regulated by different promoters. HEK293 cells were infected at an infectious dose of 1000 multiplicity of infection (MOI), 3 replicates per virus. 48h after viral infection of the cells, the level of Fluc expression in the cells was measured using the Bright-Glo luciferase assay System (Promega, USA), and the results are expressed in relative light intensity units (RLU). CA, transducing rAAVDJ-CA-Fluc virus cells; CAS, transducing rAAVDJ-CAS-Fluc viral cells; CAR, transducing rAAVDJ-CAR-Fluc viral cells; CAT, transducing rAAVDJ-CAT-Fluc virus cells; CAP, transduction of rAAVDJ-CAP-Fluc virus cells.

FIG. 20 comparison of expression efficiency of different promoters in GM03813 cells. The expression efficiency of different promoters is compared by taking the firefly luciferase Fluc as a reporter gene. Packaging to obtain the AAVDJ virus carrying Fluc expression frames regulated by different promoters. GM03813 cells were infected at an infective dose of 1000 multiplicity of infection (MOI), 3 multiple wells per virus. 48h after viral infection of the cells, the level of Fluc expression in the cells was measured using the Bright-Glo luciferase assay System (Promega, USA), and the results are expressed in relative light intensity units (RLU). CA, transducing rAAVDJ-CA-Fluc virus cells; CAS, transducing rAAVDJ-CAS-Fluc viral cells; CAR, transducing rAAVDJ-CAR-Fluc viral cells; CAT, transducing rAAVDJ-CAT-Fluc virus cells; CAP, transduction of rAAVDJ-CAP-Fluc virus cells.

FIG. 21 expression of different promoters in the liver of C57BL/6J miceAnd comparing the results of the efficiency. The expression efficiency of different promoters is compared by taking the firefly luciferase Fluc as a reporter gene. Packaging to prepare the AAV9 virus carrying Fluc expression cassettes regulated by different promoters. At 1 × 10¹²The dose of vg (viral genome)/v was injected via tail vein into C57BL/6J mice, 5 mice per virus. After 4 weeks of virus injection, mice were sacrificed, livers were separated, total tissue proteins were extracted using a total tissue cell protein extraction kit (Beijing prilley Gene technology Co., Ltd.), and the protein concentration of the extracted total proteins was measured using a protein quantification kit (BCA method) (Beijing prilley Gene technology Co., Ltd.). According to the concentration measured, the total protein was diluted to 0.5. mu.g/. mu.L, and 20. mu.L of the protein solution was used to measure the level of Fluc expression in cells using the Bright-Glo luciferase assay System (Promega, USA), and the measurement results were expressed in relative light intensity units (RLU). CA, transduction of rAAV9-CA-Fluc virus mice; CAS, transduction of rAAV9-CAS-Fluc virus mice; CAR, rAAV9-CAR-Fluc virus transduced mice; CAT, transduction rAAV9-CAT-Fluc virus mouse; CAP, transduction of rAAV9-CAP-Fluc virus mice.

FIG. 22 shows the comparison of the expression efficiency of different promoters in the brain of C57BL/6J mouse. The expression efficiency of different promoters is compared by taking the firefly luciferase Fluc as a reporter gene. Packaging to prepare the AAV9 virus carrying Fluc expression cassettes regulated by different promoters. At 1 × 10¹²The dose of vg (viral genome)/v was injected via tail vein into C57BL/6J mice, 5 mice per virus. After 4 weeks of virus injection, mice were sacrificed, brain tissues were isolated, total tissue proteins were extracted using a total tissue cell protein extraction kit (Beijing prilley Gene technology Co., Ltd.), and the protein concentration of the extracted total proteins was measured using a protein quantification kit (BCA method) (Beijing prilley Gene technology Co., Ltd.). According to the concentration measured, the total protein was diluted to 0.5. mu.g/. mu.L, and 20. mu.L of the protein solution was used to measure the level of Fluc expression in cells using the Bright-Glo luciferase assay System (Promega, USA), and the measurement results were expressed in relative light intensity units (RLU). CA, transduction of rAAV9-CA-Fluc virus mice; CAS, transduction of rAAV9-CAS-Fluc VirusA mouse; CAR, rAAV9-CAR-Fluc virus transduced mice; CAT, transduction rAAV9-CAT-Fluc virus mouse; CAP, transduction of rAAV9-CAP-Fluc virus mice.

FIG. 23 injection of recombinant AAV viruses carrying SMN1 or coSMN1 gene expression cassettes to prolong survival in SMA model mice. 6 different recombinant AAV viruses (AAV 9-CA-Fluc, AAV9-CAR-Fluc, scAAV9-CA-SMN1, scAAV9-CA-cosMN1, scAAV9-CAR-SMN1, scAAV9-CAR-cosMN 1) at 5X 10¹³SMA model mice (SMN 2) were injected tail vein at a dose of vg/kg (viral genome, vg)^+/+, SMNΔ7^+/+, smn^-/-Mice) with AAV9-CA-Fluc and AAV9-CAR-Fluc as control viruses, 5 SMA model mice were injected for each virus, the SMA mice age at 1 day of age at the time of injection. After virus injection, the survival of the mice was recorded. WT, C57BL/6J wild-type control mice; KO-AAV9-CA-Fluc, an SMA model mouse injected with AAV9-CA-Fluc virus; KO-AAV9-CAR-Fluc, SMA model mice injected with AAV9-CAR-Fluc virus; KO-AAV9-CA-SMN1, SMA model mouse injected with scAAV9-CA-SMN1 virus; KO-AAV9-CA-cosMN1, SMA model mouse injected with scAAV9-CA-cosMN1 virus; KO-AAV9-CAR-SMN1, SMA model mice injected with scAAV9-CAR-SMN1 virus; KO-AAV9-CAR-cosMN1, SMA model mouse injected with scAAV9-CAR-cosMN1 virus.

FIG. 24 shows body weight changes in SMA model mice injected with recombinant AAV virus carrying SMN1 or coSMN1 gene expression cassettes. 6 different recombinant AAV viruses (AAV 9-CA-Fluc, AAV9-CAR-Fluc, scAAV9-CA-SMN1, scAAV9-CA-cosMN1, scAAV9-CAR-SMN1, scAAV9-CAR-cosMN 1) at 5X 10¹³SMA model mice (SMN 2) were injected tail vein at a dose of vg/kg (viral genome, vg)^+/+, SMNΔ7^+/+, smn^-/-Mice) with AAV9-CA-Fluc and AAV9-CAR-Fluc as control viruses, 5 SMA model mice were injected for each virus, the SMA mice age at 1 day of age at the time of injection. After injection of the virus, the body weight changes of the mice were recorded. WT, C57BL/6J wild-type control mice; KO-AAV9-CA-Fluc, an SMA model mouse injected with AAV9-CA-Fluc virus; KO-AAV9-CAR-Fluc, SMA model mice injected with AAV9-CAR-Fluc virus; KO-AAV9-CA-SMN1, SMA model mouse injected with scAAV9-CA-SMN1 virus; KO-AAV9-CA-cosMN1,SMA model mice injected with scAAV9-CA-coSMN1 virus; KO-AAV9-CAR-SMN1, SMA model mice injected with scAAV9-CAR-SMN1 virus; KO-AAV9-CAR-cosMN1, SMA model mouse injected with scAAV9-CAR-cosMN1 virus.

FIG. 25 Effect of the addition of miR-122 target sequences to the SMN1 or coSMN1 gene expression cassettes on survival of SMA model mice. 4 different recombinant AAV viruses (scAAV 9-CAR-SMN1, scAAV9-CAR-cosMN1, scAAV9-CAR-SMN1-122T, scAAV9-CAR-cosMN 1-122T) at 5X 10¹³SMA model mice (SMN 2) were injected tail vein at a dose of vg/kg (viral genome, vg)^+/+, SMNΔ7^+/+, smn^-/-Mouse), wherein the gene expression cassette of the scAAV9-CAR-SMN1-122T, scAAV9-CAR-coSMN1-122T virus SMN1 or coSMN1 contains a single, fully base-complementary human miR-122 target sequence, 5 mice are injected with each virus, and the age of the SMA mice at the time of injection is 1 day old. After virus injection, the survival of the mice was recorded. WT, C57BL/6J wild-type control mice; KO-AAV9-CAR-SMN1, SMA model mice injected with scAAV9-CAR-SMN1 virus; KO-AAV9-CAR-coSMN1, SMA model mouse injected with scAAV9-CAR-coSMN1 virus; KO-AAV9-CAR-SMN1-122T, SMA model mice injected with scAAV9-CAR-SMN1-122T virus; KO-AAV9-CAR-cosMN1-122T, SMA model mouse injected with scAAV9-CAR-cosMN1-122T virus.

FIG. 26 the effect of the addition of miR-122 target sequences to the expression cassette of the SMN1 or coSMN1 gene on body weight changes in SMA model mice. 4 different recombinant AAV viruses (scAAV 9-CAR-SMN1, scAAV9-CAR-cosMN1, scAAV9-CAR-SMN1-122T, scAAV9-CAR-cosMN 1-122T) at 5X 10¹³SMA model mice (SMN 2) were injected tail vein at a dose of vg/kg (viral genome, vg)^+/+, SMNΔ7^+/+, smn^-/-Mouse), wherein the gene expression cassette of the scAAV9-CAR-SMN1-122T, scAAV9-CAR-coSMN1-122T virus SMN1 or coSMN1 contains a single, fully base-complementary human miR-122 target sequence, 5 mice are injected with each virus, and the age of the SMA mice at the time of injection is 1 day old. After injection of the virus, the body weight change of the mice was recorded. WT, C57BL/6J wild-type control mice; KO-AAV9-CAR-SMN1, SMA model mice injected with scAAV9-CAR-SMN1 virus; KO-AAV9-CAR-coSMN1, scAAV9-CAR-coSMN1 injectionSMA model mice for virus; KO-AAV9-CAR-SMN1-122T, SMA model mice injected with scAAV9-CAR-SMN1-122T virus; KO-AAV9-CAR-cosMN1-122T, SMA model mouse injected with scAAV9-CAR-cosMN1-122T virus.

FIG. 27 changes in expression levels of CoSMN1 in SMA mice after transduction of a virus containing the miR-122 target sequence. scAAV9-CAR-cosMN1 and scAAV9-CAR-cosMN1-122T recombinant AAV viruses at 5X 10¹³SMA model mice (SMN 2) were injected tail vein at a dose of vg/kg (viral genome, vg)^+/+, SMNΔ7^+/+, smn^-/-Mice) in which the scAAV9-CAR-cosMN1-122T virus has a single, fully base-complementary human miR-122 target sequence in the cosMN1 gene expression cassette, 5 mice were injected for each virus, and the age of the SMA mice at the time of injection was 1 day old. After 3 months of virus injection, tissues of heart, liver, skeletal muscle, lung, spleen, kidney and brain were isolated. Extracting total RNA of the tissues, detecting the copy number of cosMN1 RNA and GAPDH RNA in the total RNA by quantitative PCR, and calculating the ratio of the copy number of cosMN1 RNA and the copy number of GAPDH RNA to express the expression level of the cosMN1 gene. KO, not injected Virus SMN2^+/+, SMNΔ7^+/+, smn^-/-A control mouse; KO-AAV9-CAR-coSMN1, SMA model mouse injected with scAAV9-CAR-coSMN1 virus; KO-AAV9-CAR-cosMN1-122T, SMA model mouse injected with scAAV9-CAR-cosMN1-122T virus.

FIG. 28 effect of different forms of ITR structural virus on survival of SMA model mice. 4 different recombinant AAV viruses (scAAV 9-CAR-cosMN1, scAAV9-CAR-cosMN1-122T, scAAV9-U-CAR-cosMN1, scAAV9-U-CAR-cosMN 1-122T) at 5X 10¹³SMA model mice (SMN 2) were injected tail vein at a dose of vg/kg (viral genome, vg)^+/+, SMNΔ7^+/+, smn^-/-Mice) 5 SMA model mice were injected for each virus, the SMA mice age at the time of injection being 1 day old. Wherein the scAAV9-CAR-coSMN1, scAAV9-CAR-coSMN1-122T virus contains the ITRs of normal AAV2 and the ITRs of normal AAV2 deleted for the D sequence (Δ ITRs); the scAAV9-U-CAR-cosMN1, scAAV9-U-CAR-cosMN1-122T viruses contain AAV2 ITRs (U-ITRs) with deletion of B-B 'and C-C' sequences, and AAV2 ITRs (Δ U-ITRs) with deletion of D, B-B 'and C-C' sequences. Injection of virusThereafter, the survival of the mice was recorded. WT, C57BL/6J wild-type control mice; KO-AAV9-CAR-coSMN1, SMA model mouse injected with scAAV9-CAR-coSMN1 virus; KO-AAV9-CAR-cosMN1-122T, SMA model mouse injected with scAAV9-CAR-cosMN1-122T virus; KO-AAV9-U-CAR-SMN1-122T, SMA model mice injected with scAAV9-U-CAR-SMN1-122T virus; KO-AAV9-U-CAR-cosMN1-122T, SMA model mice injected with scAAV9-U-CAR-cosMN1-122T virus.

FIG. 29 Effect of different forms of ITR structural viruses on weight change in SMA model mice. 4 different recombinant AAV viruses (scAAV 9-CAR-cosMN1, scAAV9-CAR-cosMN1-122T, scAAV9-U-CAR-cosMN1, scAAV9-U-CAR-cosMN 1-122T) at 5X 10¹³SMA model mice (SMN 2) were injected tail vein at a dose of vg/kg (viral genome, vg)^+/+, SMNΔ7^+/+, smn^-/-Mice) 5 SMA model mice were injected for each virus, the SMA mice age at the time of injection being 1 day old. Wherein the scAAV9-CAR-coSMN1, scAAV9-CAR-coSMN1-122T virus contains the ITRs of normal AAV2 and the ITRs of normal AAV2 deleted for the D sequence (Δ ITRs); the scAAV9-U-CAR-cosMN1, scAAV9-U-CAR-cosMN1-122T viruses contain AAV2 ITRs (U-ITRs) with deletion of B-B 'and C-C' sequences, and AAV2 ITRs (Δ U-ITRs) with deletion of D, B-B 'and C-C' sequences. After injection of the virus, the body weight change of the mice was recorded. WT, C57BL/6J wild-type control mice; KO-AAV9-CAR-coSMN1, SMA model mouse injected with scAAV9-CAR-coSMN1 virus; KO-AAV9-CAR-cosMN1-122T, SMA model mouse injected with scAAV9-CAR-cosMN1-122T virus; KO-AAV9-U-CAR-SMN1-122T, SMA model mice injected with scAAV9-U-CAR-SMN1-122T virus; KO-AAV9-U-CAR-cosMN1-122T, SMA model mice injected with scAAV9-U-CAR-cosMN1-122T virus.

FIG. 30 changes in expression levels of CoSMN1 in SMA mice following transduction of different forms of ITR structural virus. 4 different recombinant AAV viruses (scAAV 9-CAR-cosMN1, scAAV9-CAR-cosMN1-122T, scAAV9-U-CAR-cosMN1, scAAV9-U-CAR-cosMN 1-122T) at 5X 10¹³SMA model mice (SMN 2) were injected tail vein at a dose of vg/kg (viral genome, vg)^+/+, SMNΔ7^+/+, smn^-/-Mice), 5 SMA model mice were injected for each virus, SMA mice at the time of injectionThe age was 1 day old. Wherein the scAAV9-CAR-coSMN1, scAAV9-CAR-coSMN1-122T virus contains the ITRs of normal AAV2 and the ITRs of normal AAV2 deleted for the D sequence (Δ ITRs); the scAAV9-U-CAR-cosMN1, scAAV9-U-CAR-cosMN1-122T viruses contain AAV2 ITRs (U-ITRs) with deletion of B-B 'and C-C' sequences, and AAV2 ITRs (Δ U-ITRs) with deletion of D, B-B 'and C-C' sequences. After 3 months of virus injection, tissues of heart, liver, skeletal muscle, lung, spleen, kidney and brain were isolated. Extracting total RNA of the tissues, detecting the copy number of cosMN1 RNA and GAPDH RNA in the total RNA by quantitative PCR, and calculating the ratio of the copy number of cosMN1 RNA and the copy number of GAPDH RNA to express the expression level of the cosMN1 gene. KO, not injected Virus SMN2^+/+, SMNΔ7^+/+, smn^-/-A control mouse; KO-AAV9-CAR-coSMN1, SMA model mouse injected with scAAV9-CAR-coSMN1 virus; KO-AAV9-CAR-cosMN1-122T, SMA model mouse injected with scAAV9-CAR-cosMN1-122T virus; KO-AAV9-U-CAR-SMN1-122T, SMA model mice injected with scAAV9-U-CAR-SMN1-122T virus; KO-AAV9-U-CAR-cosMN1-122T, SMA model mice injected with scAAV9-U-CAR-cosMN1-122T virus.

Figure 31 effect of different injection regimens on survival in SMA model mice. 2 different recombinant AAV viruses (scAAV 9-CAR-cosMN1, scAAV9-CAR-cosMN 1-122T) were injected into SMA model mice (SMN 2)^+/+, SMNΔ7^+/+, smn^-/-Mice) 5 SMA model mice were injected per virus per injection mode, with SMA mice age 1 day old at the time of injection. Wherein the intravenous injection mode (IV) has an injection dosage of 5 × 10¹³vg/kg (viral genome, vg), and the injection dose of the intrathecal Injection (IT) is 1X 10¹³vg/kg (viral genome, vg), the injection dose of intracerebroventricular injection mode (ICV) is 1X 10¹³vg/kg (viral genome, vg). After virus injection, the survival of the mice was recorded. WT, C57BL/6J wild-type control mice; KO-AAV9-CAR-cosMN1 (IV), an SMA model mouse injected intravenously with scAAV9-CAR-cosMN1 virus; KO-AAV9-CAR-cosMN1-122T (IV), an SMA model mouse injected intravenously with scAAV9-CAR-cosMN1-122T virus; KO-AAV9-CAR-cosMN1 (IT), intrathecally injecting scAAV9-CASMA model mouse of R-coSMN1 virus; KO-AAV9-CAR-cosMN1-122T (IT), an SMA model mouse intrathecally injected with scAAV9-CAR-cosMN1-122T virus; KO-AAV9-CAR-cosMN1 (ICV), an SMA model mouse injected intracerebroventricularly with scAAV9-CAR-cosMN1 virus; KO-AAV9-CAR-cosMN1-122T (ICV), an SMA model mouse injected intracerebroventricularly with scAAV9-CAR-cosMN1-122T virus.

FIG. 32 Effect of different injection regimens on weight change in SMA model mice. 2 different recombinant AAV viruses (scAAV 9-CAR-cosMN1, scAAV9-CAR-cosMN 1-122T) were injected into SMA model mice (SMN 2)^+/+, SMNΔ7^+/+, smn^-/-Mice) 5 SMA model mice were injected per virus per injection mode, with SMA mice age 1 day old at the time of injection. Wherein the intravenous injection mode (IV) has an injection dosage of 5 × 10¹³vg/kg (viral genome, vg), and the injection dose of the intrathecal Injection (IT) is 1X 10¹³vg/kg (viral genome, vg), the injection dose of intracerebroventricular injection mode (ICV) is 1X 10¹³vg/kg (viral genome, vg). After injection of the virus, the body weight of the mice was recorded. WT, C57BL/6J wild-type control mice; KO-AAV9-CAR-cosMN1 (IV), an SMA model mouse injected intravenously with scAAV9-CAR-cosMN1 virus; KO-AAV9-CAR-cosMN1-122T (IV), an SMA model mouse injected intravenously with scAAV9-CAR-cosMN1-122T virus; KO-AAV9-CAR-cosMN1 (IT), an SMA model mouse injected intrathecally with scAAV9-CAR-cosMN1 virus; KO-AAV9-CAR-cosMN1-122T (IT), an SMA model mouse intrathecally injected with scAAV9-CAR-cosMN1-122T virus; KO-AAV9-CAR-cosMN1 (ICV), an SMA model mouse injected intracerebroventricularly with scAAV9-CAR-cosMN1 virus; KO-AAV9-CAR-cosMN1-122T (ICV), an SMA model mouse injected intracerebroventricularly with scAAV9-CAR-cosMN1-122T virus.

FIG. 33 comparison of the expression levels of CoSMN1 in SMA mice under different injection modes. 2 different recombinant AAV viruses (scAAV 9-CAR-cosMN1, scAAV9-CAR-cosMN 1-122T) were injected into SMA model mice (SMN 2)^+/+, SMNΔ7^+/+, smn^-/-Mouse), each virus injection formulaFormula 5 SMA model mice were injected, and the age of SMA mice at the time of injection was 1 day old. Wherein the intravenous injection mode (IV) has an injection dosage of 5 × 10¹³vg/kg (viral genome, vg), and the injection dose of the intrathecal Injection (IT) is 1X 10¹³vg/kg (viral genome, vg), the injection dose of intracerebroventricular injection mode (ICV) is 1X 10¹³vg/kg (viral genome, vg). After 3 months of virus injection, tissues of heart, liver, skeletal muscle, lung, spleen, kidney and brain were isolated. Extracting total RNA of the tissues, detecting the copy number of cosMN1 RNA and GAPDH RNA in the total RNA by quantitative PCR, and calculating the ratio of the copy number of cosMN1 RNA and the copy number of GAPDH RNA to express the expression level of the cosMN1 gene. KO, not injected Virus SMN2^+/+, SMNΔ7^+/+, smn^-/-A control mouse; KO-AAV9-CAR-cosMN1 (IV), an SMA model mouse injected intravenously with scAAV9-CAR-cosMN1 virus; KO-AAV9-CAR-cosMN1-122T (IV), an SMA model mouse injected intravenously with scAAV9-CAR-cosMN1-122T virus; KO-AAV9-CAR-cosMN1 (IT), an SMA model mouse injected intrathecally with scAAV9-CAR-cosMN1 virus; KO-AAV9-CAR-cosMN1-122T (IT), an SMA model mouse intrathecally injected with scAAV9-CAR-cosMN1-122T virus; KO-AAV9-CAR-cosMN1 (ICV), an SMA model mouse injected intracerebroventricularly with scAAV9-CAR-cosMN1 virus; KO-AAV9-CAR-cosMN1-122T (ICV), an SMA model mouse injected intracerebroventricularly with scAAV9-CAR-cosMN1-122T virus.

FIG. 34 Effect of different AAV vector serotypes on survival of SMA model mice upon intravenous injection. 3 different recombinant AAV viruses (scAAV 5-CAR-cosMN1, scAAV9-CAR-cosMN1, scAAVrh10-CAR-cosMN 1) at 5X 10¹³Doses of vg/kg were injected intravenously into SMA model mice (SMN 2)^+/+, SMNΔ7^+/+, smn^-/-Mice) 5 SMA model mice were injected for each virus, the SMA mice age at the time of injection being 1 day old. After virus injection, the survival of the mice was recorded. WT, C57BL/6J wild-type control mice; KO-AAV5-CAR-cosMN1, an SMA model mouse injected intravenously with scAAV5-CAR-cosMN1 virus; KO-AAV9-CAR-cosMN1, SMA model mouse injected intravenously with scAAV9-CAR-cosMN1 virus(ii) a KO-AAVrh10-CAR-cosMN1, an SMA model mouse injected intravenously with scAAV9-CAR-cosMN1 virus.

FIG. 35 Effect of different AAV vector serotypes on weight change in SMA model mice following intravenous injection. 3 different recombinant AAV viruses (scAAV 5-CAR-cosMN1, scAAV9-CAR-cosMN1, scAAVrh10-CAR-cosMN 1) at 5X 10¹³Doses of vg/kg were injected intravenously into SMA model mice (SMN 2)^+/+, SMNΔ7^+/+, smn^-/-Mice) 5 SMA model mice were injected for each virus, the SMA mice age at the time of injection being 1 day old. After injection of the virus, the body weight of the mice was recorded. WT, C57BL/6J wild-type control mice; KO-AAV5-CAR-cosMN1, an SMA model mouse injected intravenously with scAAV5-CAR-cosMN1 virus; KO-AAV9-CAR-cosMN1, an SMA model mouse injected intravenously with scAAV9-CAR-cosMN1 virus; KO-AAVrh10-CAR-cosMN1, an SMA model mouse injected intravenously with scAAV9-CAR-cosMN1 virus.

FIG. 36 Effect of different AAV vector serotypes on survival in SMA model mice following intrathecal injection. 3 different recombinant AAV viruses (scAAV 5-CAR-cosMN1, scAAV9-CAR-cosMN1, scAAVrh10-CAR-cosMN 1) at 1X 10¹³Doses of vg/kg (vral genome, vg) were injected intrathecally into SMA model mice (SMN 2)^+/+, SMNΔ7^+/+, smn^-/-Mice) 5 SMA model mice were injected for each virus, the SMA mice age at the time of injection being 1 day old. After virus injection, the survival of the mice was recorded. WT, C57BL/6J wild-type control mice; KO-AAV5-CAR-cosMN1, a SMA model mouse intrathecally injected with scAAV5-CAR-cosMN1 virus; KO-AAV9-CAR-cosMN1, a SMA model mouse intrathecally injected with scAAV9-CAR-cosMN1 virus; KO-AAVrh10-CAR-cosMN1, SMA model mouse intrathecally injected with scAAV9-CAR-cosMN1 virus.

Figure 37 the effect of different AAV vector serotypes on body weight change in SMA model mice following intrathecal injection. 3 different recombinant AAV viruses (scAAV 5-CAR-cosMN1, scAAV9-CAR-cosMN1, scAAVrh10-CAR-cosMN 1) at 1X 10¹³Doses of vg/kg (vral genome, vg) were injected intrathecally into SMA model mice (SMN 2)^+/+, SMNΔ7^+/+, smn^-/-Mice) 5 SMA model mice were injected for each virus, the SMA mice age at the time of injection being 1 day old. After injection of the virus, the body weight of the mice was recorded. WT, C57BL/6J wild-type control mice; KO-AAV5-CAR-cosMN1, a SMA model mouse intrathecally injected with scAAV5-CAR-cosMN1 virus; KO-AAV9-CAR-cosMN1, a SMA model mouse intrathecally injected with scAAV9-CAR-cosMN1 virus; KO-AAVrh10-CAR-cosMN1, SMA model mouse intrathecally injected with scAAV9-CAR-cosMN1 virus.

FIG. 38 Effect of different AAV vector serotypes on survival in SMA model mice following intracerebroventricular injection. 3 different recombinant AAV viruses (scAAV 5-CAR-cosMN1, scAAV9-CAR-cosMN1, scAAVrh10-CAR-cosMN 1) at 1X 10¹³Doses of vg/kg were injected intracerebroventricularly into SMA model mice (SMN 2)^+/+, SMNΔ7^+/+, smn^-/-Mice) 5 SMA model mice were injected for each virus, the SMA mice age at the time of injection being 1 day old. After virus injection, the survival of the mice was recorded. WT, C57BL/6J wild-type control mice; KO-AAV5-CAR-cosMN1, an SMA model mouse injected intracerebroventricularly with scAAV5-CAR-cosMN1 virus; KO-AAV9-CAR-cosMN1, an SMA model mouse injected intracerebroventricularly with scAAV9-CAR-cosMN1 virus; KO-AAVrh10-CAR-cosMN1, an SMA model mouse injected intracerebroventricularly with scAAV9-CAR-cosMN1 virus.

FIG. 39 the effect of different AAV vector serotypes on weight change in SMA model mice following intracerebroventricular injection. 3 different recombinant AAV viruses (scAAV 5-CAR-cosMN1, scAAV9-CAR-cosMN1, scAAVrh10-CAR-cosMN 1) at 1X 10¹³Doses of vg/kg were injected intracerebroventricularly into SMA model mice (SMN 2)^+/+, SMNΔ7^+/+, smn^-/-Mice) 5 SMA model mice were injected for each virus, the SMA mice age at the time of injection being 1 day old. After injection of the virus, the body weight of the mice was recorded. WT, C57BL/6J wild-type control mice; KO-AAV5-CAR-cosMN1, an SMA model mouse injected intracerebroventricularly with scAAV5-CAR-cosMN1 virus; KO-AAV9-CAR-cosMN1, an SMA model mouse injected intracerebroventricularly with scAAV9-CAR-cosMN1 virus; KO-AAVrh10-CAR-cosMN1, an SMA model mouse injected intracerebroventricularly with scAAV9-CAR-cosMN1 virus.

Detailed Description

The invention discloses a series of recombinant adeno-associated viruses carrying artificially designed SMN1 gene expression cassettes, which comprises the design, minipreparation and functional verification of the viruses. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention. In which, unless otherwise specified, the various reagents mentioned in the examples are commercially available.

The invention is further illustrated by the following examples:

example 1 plasmid vector construction

(1) Promoter design Synthesis

Reference (Niwa H, et al, Gene 1991;108: 193-200.) is based on the CAG promoter (consisting of CMV enhancer and chicken beta-actin promoter) in the mammalian expression vector pCAGGS vector (GenBank: LT 727518.1), and in view of the length of the sequence, a partial sequence of the chicken beta-actin promoter in the CAG promoter is deleted to obtain a truncated CAG promoter, which is designated as CA promoter. Next, a partial sequence of the 5 'untranslated region of human SMN1 gene mRNA (GenBank: NM-001297715.1) was introduced at the 3' end of the CA promoter sequence to obtain a name of CAS promoter. The intron sequences 449 to 532 in the human RNA polymerase II 14.5kDa subunit gene (GenBank: Z23102.1) were introduced into the 3' end of the CA promoter sequence to obtain the promoter named CAT promoter. An intron sequence from 62804 th to 62890 th in a human TATA box binding protein related factor 1 gene (GenBank: NG-012771.2) is introduced at the 3' end of the CA promoter sequence to obtain the promoter named CAR. A partial sequence of the 5 'untranslated region of human phosphoglycerate kinase gene mRNA (GenBank: M34017.1) was introduced at the 3' end of the CA promoter sequence to obtain the promoter named CAP. The CAS promoter, CAT promoter, CAR promoter, CAP promoter and CA promoter are sent to Nanjing Kingsler Biotech company for synthesis, and the sequence information is CAS promoter (SEQ ID No. 1), CAT promoter (SEQ ID No. 2), CAR promoter (SEQ ID No. 3), CAP promoter (SEQ ID No. 4) and CA promoter (SEQ ID No. 5). For the convenience of subsequent cloning, the promoter is designed to introduce XhoI (5 'CTCGAG 3') cutting site at the 5 'end and KpnI (5' GGTACC3 ') cutting site at the 3' end during the sequence synthesis. The synthetic promoter sequences were cloned into pUC57 simple vectors (Nanjing Kingsrei Biotech) and designated pUC57-CAS (containing the CAS promoter), pUC57-CAT (containing the CAT promoter), pUC57-CAR (containing the CAR promoter), pUC57-CAP (containing the CAP promoter) and pUC57-CA (containing the CA promoter), respectively.

(2) Fluc reporter Gene vector construction

Based on pAAV2neo (FIG. 1) stored in the company, firefly luciferase gene Fluc was inserted between the KpnI and BglII cleavage sites of the pAAV2neo vector to obtain the pAAV2neo-Fluc vector (FIG. 2). Specifically, pGL4.14[ luc2/Hygro ] vector (purchased from Promega, USA) is used as a template, and two primers (Fluc-F and Fluc-R) are designed for PCR amplification to obtain a sequence fragment containing a Fluc coding region. And (3) carrying out double enzyme digestion and PCR amplification on KpnI and BglII to obtain a fragment, and recovering for later use. The pAAV2neo vector is digested with KpnI and BglII by double digestion, linearized and recovered for later use. And connecting the two recovered fragments, transforming E.coli JM109 competent cells (purchased from Dalibao organisms), selecting colonies, extracting plasmids, and carrying out enzyme digestion identification to obtain the pAAV2neo-Fluc vector.

Fluc-F 5’ataggtaccgccaccatggaagatgcc3’ (SEQ ID No.16)

Fluc-R 5’attagatctttacacggcgatcttgcc3’ (SEQ ID No.17)

The pAAV2neo-Fluc vector is subjected to double digestion by XhoI and KpnI to generate two fragments of 7808bp and 778bp, and the vector fragment with the length of 7808bp is recovered for later use. XhoI and KpnI double digestion separately digested pUC57-CAS (containing CAS promoter), pUC57-CAT (containing CAT promoter), pUC57-CAR (containing CAR promoter), pUC57-CAP (containing CAP promoter) and pUC57-CA (containing CA promoter) vectors, generated about 600bp and 2.7kb two fragments, and recovered about 600bp long promoter fragment. Respectively connecting the recovered 7808bp vector fragment and the recovered promoter fragment, transforming E.coli JM109 competent cells (purchased from Dalibao), picking colonies, extracting plasmids, and carrying out enzyme digestion identification to respectively obtain a pAAV2neo-CAS-Fluc vector (figure 3), a pAAV2neo-CAT-Fluc vector (figure 4), a pAAV2neo-CAR-Fluc vector (figure 5), a pAAV2neo-CAP-Fluc vector (figure 6) and a pAAV2neo-CA-Fluc vector (figure 7).

Based on the 3' ITR sequence in AAV2 genome (GenBank number AF 043303), the trs sequence and D sequence in the ITR sequence were deleted according to the literature report (Wang Z, et al Gene ther 2003;10: 2105-2111.) to obtain the delta ITR sequence (SEQ ID No. 6). For the convenience of cloning operation, a sequence between 1392-2127bp (a sequence between ITR and SwaI enzyme cutting site close to BGH polyA) in the pAAV2neo vector is fused with the delta ITR sequence to obtain a fusion sequence delta ITR-BS (SEQ ID No. 7). BamHI and SwaI restriction sites were added to both ends of the Δ ITR-BS fusion sequence, respectively, and then synthesized by Nanjing King Shirui Biotechnology Co., Ltd, and cloned into pUC57 simple vector to obtain pUC57- Δ ITR-BS. And carrying out double digestion on the pUC 57-delta ITR-BS vector, the pAAV2neo-CAR-Fluc vector and the pAAV2neo-CA-Fluc vector by BamHI and SwaI respectively, and recovering a delta ITR-BS fragment, an ITR sequence cut pAAV2neo-CAR-Fluc vector fragment and an ITR sequence cut pAAV2neo-CA-Fluc vector fragment. The recovered delta ITR-BS fragments are respectively connected with the pAAV2neo-CAR-Fluc vector fragment with the cut-off ITR sequence or the pAAV2neo-CA-Fluc vector fragment with the cut-off ITR sequence, E.coli JM109 competent cells (Botrytis, Dalian) are transformed, and the pscAAV-CA-Fluc vector (figure 8) and the pscAAV-CAR-Fluc vector (figure 9) are obtained through screening and identification.

Based on the 3 ' ITR sequence in AAV2 genome (GenBank number AF 043303), the U-ITR sequence (SEQ ID No. 8) was obtained by deleting the B-B ' and C-C ' sequences in the ITR sequence according to the patent (patent application No. CN 201510931560.3.) and the literature (Zhou Q, et al Sci Rep.2017; 7(1): 5432.). For the convenience of cloning operation, the sequence between 1392-2127bp (the sequence between ITR and SwaI enzyme cutting site close to BGH polyA) in the pAAV2neo vector is fused with the U-ITR sequence to obtain a fusion sequence U-ITR-BS (SEQ ID No. 9). BamHI and SwaI restriction sites were added to both ends of the U-ITR-BS fusion sequence, respectively, and then synthesized by Nanjing Kingsler Biotechnology Ltd, and cloned into pUC57 simple vector to obtain pUC 57-U-ITR-BS. Further deletion of the trs sequence and the D sequence in the U-ITR sequence gave a.DELTA.U-ITR sequence (SEQ ID No. 10). To maintain the consistency of the vector backbone sequence and ease of cloning, the sequence "5'-gctagaacaacaa-3'" was introduced at the 3 ' end of the Δ U-ITR sequence, resulting in a Δ U-ITR-MX sequence (SEQ ID No. 11). XhoI and MfeI restriction sites are respectively added at two ends of the delta U-ITR-MX sequence, then the mixture is synthesized by Nanjing Kingsry Biotechnology limited company and cloned into a pUC57 simple vector to obtain pUC 57-delta U-ITR-MX.

And carrying out double digestion on the pUC57-U-ITR-BS vector and the pAAV2neo-CAR-Fluc vector by BamHI and SwaI respectively, recovering a U-ITR-BS fragment and an ITR sequence cut pAAV2neo-CAR-Fluc vector fragment. And connecting the recovered U-ITR-BS fragment with the pAAV2neo-CAR-Fluc vector fragment with an ITR sequence cut off, transforming E.coli JM109 competent cells (Baozoite, Dalian), and screening and identifying to obtain the pscAAV-U-CAR-Fluc-pre vector.

And (3) carrying out double digestion on pUC 57-delta U-ITR-MX vector and pscAAV-U-CAR-Fluc-pre vector by MfeI and XhoI, recovering a delta U-ITR-MX fragment and cutting off a pscAAV-U-CAR-Fluc-pre vector fragment with an ITR sequence. The recovered delta U-ITR-MX fragment was ligated with a pscAAV-U-CAR-Fluc-pre vector fragment from which the ITR sequence was excised, E.coli JM109 competent cells (Takara Bio, Dalian) were transformed, and the vector was screened and identified to obtain a pscAAV-U-CAR-Fluc vector (FIG. 10).

(3) SMN1 and coSMN1 gene vector

The NCBI GenBank database (https:// www.ncbi.nlm.nih.gov/gene /) was searched for the SMN1 gene mRNA sequence (GenBank: NM-000344.3). The coding sequence of the SMN1 protein was analyzed from the mRNA sequence, and a "5 ' -GCCACC-3 '" sequence was added to the 5 ' end of the coding sequence to obtain the SMN1 sequence (SEQ ID No. 12). The SMN1 protein coding region sequence was codon optimized and a "5 ' -GCCACC-3 '" sequence was added to the 5 ' end of the optimized sequence to give a COSMN1 sequence (SEQ ID No. 13). The SMN1 and COSMN1 sequences were sent to Nanjing Kinsrui Biotech, Inc. for synthesis. For the convenience of subsequent cloning, the 5 'end of the designed promoter is introduced with KpnI (5' GGTACC3 ') cutting site during sequence synthesis, and the 3' end is introduced with BglII (5 'AGATCT 3') cutting site. The synthetic promoter sequences were cloned into pUC57 simple vectors (Biotech, King-Story, Nanjing) and designated pUC57-SMN1 (containing the SMN1 sequence) and pUC57-coSMN1 (containing the coSMN1 sequence), respectively.

The pUC57-SMN1 vector was digested with KpnI and BglII to generate two fragments of about 900bp and 2.7kb in length, and the SMN1 sequence fragment of about 900bp in length was recovered. The psCAAV-CA-Fluc vector (FIG. 8) and the psCAAV-CAR-Fluc vector (FIG. 9) were digested with KpnI and BglII, respectively, to generate two fragments of about 6.7kb and 1.7kb, respectively, and the 6.7kb fragment was recovered. And respectively connecting the SMN1 sequence fragments obtained by recovery with 6.7kb fragments recovered by digestion of a pscAAV2neo-CA-Fluc vector or digestion of the pscAAV2neo-CAR-Fluc vector, and identifying to obtain a pscAAV-CA-SMN1 vector (figure 11) and a pscAAV-CAR-SMN1 vector (figure 13).

The pUC57-coSMN1 vector was digested with KpnI and BglII to generate two fragments of about 900bp and 2.7kb in length, and a fragment of about 900bp of coSMN1 sequence was recovered. The psCAAV-CA-Fluc vector (FIG. 8), the psCAAV-CAR-Fluc vector (FIG. 9), and the psCAAV-U-CAR-Fluc vector (FIG. 10) were digested with KpnI and BglII, respectively, to generate two fragments of about 6.7kb and 1.7kb, respectively, and the fragment of 6.7kb was recovered. And respectively connecting the SMN1 sequence fragments obtained by recovery with 6.7kb fragments recovered by digestion and recovery of a pscAAV2neo-CA-Fluc vector or digestion and recovery of a pscAAV2neo-CAR-Fluc vector or digestion and recovery of a pscAAV-U-CAR-Fluc vector, and identifying to obtain a pscAAV-CA-cosMN1 vector (figure 12), a pscAAV-CAR-cosMN1 vector (figure 14) and a pscAAV-U-CAR-cosMN1 vector (figure 15).

(4) Construction of miR-122-containing target sequence vector

A single target sequence completely complementary to human miR-122 is added to the 3' end of the SMN1 sequence (SEQ ID No. 12) to obtain an SMN1-122T sequence (SEQ ID No. 14). The 3' end of the sequence of cosMN1 (SEQ ID No. 13) was added with a single target sequence fully complementary to human miR-122 to obtain a sequence of cosMN1-122T (SEQ ID No. 15). The SMN1-122T sequence and the coSMN1-122T sequence were sent to Nanjing Kingsry Biotech, Inc. for synthesis. For the convenience of subsequent cloning, the 5 'end of the designed promoter is introduced with KpnI (5' GGTACC3 ') cutting site during sequence synthesis, and the 3' end is introduced with BglII (5 'AGATCT 3') cutting site. The synthetic promoter sequences were cloned into pUC57 simple vectors (Biotech, King-Story, Nanjing) and designated pUC57-SMN1-122T (containing SMN1-122T sequences) and pUC57-coSMN1-122T (containing coSMN1-122T sequences), respectively.

The pUC57-SMN1-122T vector was digested with KpnI and BglII by double digestion to generate two fragments of about 900bp and 2.7kb in length, and the SMN1-122T sequence fragment of about 900bp in length was recovered. The pscAAV-CAR-Fluc vector (FIG. 9) was digested with KpnI and BglII by double digestion, respectively, to generate two fragments of about 6.7kb and 1.7kb, respectively, and the 6.7kb fragment was recovered. The recovered SMN1-122T sequence fragment was ligated with the recovered 6.7kb fragment by digestion with the pscAAV2neo-CAR-Fluc vector to identify the pscAAV-CAR-SMN1-122T vector (FIG. 16).

The pUC57-coSMN1-122T vector was digested with KpnI and BglII to generate two fragments of about 900bp and 2.7kb in length, and the fragment of the coSMN1-122T sequence of about 900bp in length was recovered. The psCAAV-CAR-Fluc vector (FIG. 9) and the psCAAV-U-CAR-Fluc vector (FIG. 10) were digested with KpnI and BglII, respectively, to generate two fragments of about 6.7kb and 1.7kb, respectively, and the 6.7kb fragment was recovered. The recovered cosMN1-122T sequence fragments are respectively connected with 6.7kb fragments recovered by digestion of a pscAAV2neo-CAR-Fluc vector or digestion of a pscAAV-U-CAR-Fluc vector, and a pscAAV-CAR-cosMN1-122T vector (figure 17) and a pscAAV-U-CAR-cosMN1-122T vector (figure 18) are identified.

Example 2 recombinant AAV Virus preparation and assay

Reference is made to the literature (Xiao X,et alj Virol, 1998, (72 (3): 2224-2232.) the AAV is obtained by packing the recombinant AAV with three plasmid packing systems and separating, purifying and packing with cesium chloride density gradient centrifugation. Briefly, AAV vector plasmids (pAAV 2neo-CA-Fluc, pAAV2neo-CAT-Fluc, pAAV2neo-CAP-Fluc, pAAV2neo-CAS-Fluc, pAAV2neo-CAR-Fluc, pscAAV-CA-SMN1, pscAAV-CA-cosMN1, pscAAV-CAR-SMN1, pscAAV-CAR-cosMN1, pscAAV-U-CAR-cosMN1, pscAAV-CAR-SMN1-122T, pscAAV-CAR-cosMN1-122T or pscAAV-U-CAR-cosMN 1-122T), helper plasmid (pHelper), and Rep and Cap protein expression plasmid (pAAV-DJ, pAAV-R2C5, pAAV-R2C9 or pAAV-R2C 10) of AAV were mixed at a molar ratio of 1:1:1, transfected with HEK293 cells, transfected with 48h, harvested and cultured supernatant, and purified by centrifugation using a calcium phosphate gradient. Packaging and purifying to obtain recombinant viruses such as AAVDJ-CA-Fluc, AAVDJ-CAS-Fluc, AAVDJ-CAT-Fluc, AAVDJ-CAR-Fluc, AAVDJ-CAP-Fluc, AAV9-CA-Fluc, AAV9-CAS-Fluc, AAV9-CAT-Fluc, AAV9-CAR-Fluc, AAV9-CAP-Fluc, scAAV9-CA-SMN1, scAAV9-CAR-SMN1, scAAV9-CA-COSMN1, scAAV 9-COSMN 1, scAAV 9-U-COSMN 1, scAAV9-CAR 1-122T, scAAV9-CA-COSMN 1-122-SMU 122T, scAAV 9-COSMN 1-122-COCAR 122T, scAAV 5-CAR-VrSMN 1 and AASMN 10-SMN 23-SMN 1.

And determining the genome titer of the prepared AAV by a quantitative PCR method. The specific process is as follows:

primers and probes for quantitative PCR detection were designed for the exact same sequences of 5 promoters (CA promoter, CAT promoter, CAs promoter, CAR promoter and CAP promoter):

CA-Q-F：5’- TGGGACTTTCCTACTTGGCA-3’ (SEQ ID NO.18)

CA-Q-R：5’- GGAGAGTGAAGCAGAACGTG-3’ (SEQ ID NO.19)

CA-Q-P：5’- ACCCATGGTCGAGGTGAGCCC-3’ (SEQ ID NO.20)

CA-Q-F and CA-Q-R are primers, and CA-Q-P is a probe. The 5 'end of the probe is marked by FAM fluorescent protein, and the 3' end is connected with BlackBerry query. Primers and probes were synthesized by thermolfisher Scientific. Specifically amplifying 89bp fragments in the same sequence of a CA promoter and a CAR promoter by taking CA-Q-F and CA-Q-R as primers, adopting a TaqMan probe combination method and 10⁸Copy number per ml pAAV2neo-CA-Fluc plasmid and 10 times gradient diluted sample thereof were used as standard, and Premix Ex Taq (Probe qPCR) reagent (Takara, Dalian, Medium-and Medium-Strand, in-vitro, in vitro, and in-vivo assays, were used to determine the amounts of the samplesCountry), a fluorescence quantitative PCR instrument (model: ABI 7500 fast, ABI) to detect viral genome titers. The procedures are described in Premix Ex Taq (Probe qPCR) reagent Specification. Methods for the treatment of viruses are described in the literature (Aurnhammer C, et al Hum Gene their methods, 2012; 23(1): 18-28.).

Example 3 promoter in vitro and in vivo evaluation experiment

(1) In vitro evaluation experiment

As the AAVDJ vector has higher transduction activity to various cells in vitro (Grimm D, et al. J Virol. 2008; 82: 5887-. HEK293 cells and GM03813 cell line were selected for in vitro evaluation of the designed promoter. Wherein the HEK293 cells are derived from human embryonic kidney cells. The GM03813 Cell line was purchased from Coriell Cell Repository, USA, and derived from a fibroblast Cell line of SMA type I patients (Coovert DD, et al. Hum Mol Genet. 1997; 6: 1205-1214.). The two cells were evaluated for expression activity of the designed promoter in cells derived from SMA patients and non-SMA patients, respectively.

HEK293 cells were seeded in 96-well cell culture plates. The prepared AAVDJ-CA-Fluc, AAVDJ-CAS-Fluc, AAVDJ-CAT-Fluc, AAVDJ-CAR-Fluc and AAVDJ-CAP-Fluc viruses infect HEK293 cells by the infection dose with the multiplicity of infection (MOI) of 1000 respectively, and each virus infects 3 multiple wells. 48h after viral infection of the cells, the level of Fluc expression in the cells was measured using the Bright-Glo luciferase assay System (Promega, USA), the results were expressed in relative light intensity units (RLU), and the procedures were described in the Bright-Glo luciferase assay System. The detection results are shown in fig. 19. From the results in FIG. 19, it can be seen that the expression level of Fluc after transduction of HEK293 cells with rAAVDJ-CAR-Fluc virus was significantly higher than that of the remaining 4 viruses (AAVDJ-CA-Fluc, AAVDJ-CAS-Fluc, AAVDJ-CAT-Fluc, and AAVDJ-CAP-Fluc). The expression level of Fluc after the other 4 viruses transduce HEK293 cells is AAVDJ-CA-Fluc > AAVDJ-CAP-Fluc > AAVDJ-CAT-Fluc > AAVDJ-CAS-Fluc from high to low.

Next, we infected the prepared AAVDJ-CA-Fluc, AAVDJ-CAS-Fluc, AAVDJ-CAT-Fluc, AAVDJ-CAR-Fluc, and AAVDJ-CAP-Fluc viruses with GM03813 cells derived from SMA1 patients at an infectious dose of multiple of infection (MOI) of 1000, each virus infecting 3 replicate wells. 48h after viral infection of the cells, the level of Fluc expression in the cells was measured using the Bright-Glo luciferase assay System (Promega, USA), and the results are expressed in relative light intensity units (RLU). The detection results are shown in fig. 20. From the results in fig. 20, it can be seen that the expression level of Fluc after 5 viruses transduced GM03813 cells was lower than that of HEK293 cells individually transduced. Of the 5 viruses, still rAAVDJ-CAR-Fluc transduced GM03813 cells with highest Fluc expression level, the other 4 viruses were AAVDJ-CAT-Fluc > AAVDJ-CAS-Fluc > AAVDJ-CA-Fluc > AAVDJ-CAP-Fluc in order from high to low, different from their high-low order in HEK293 cells.

In conclusion, the expression efficiency of the CAR promoter is the highest in HEK293 cells and GM03813 cells, and the CAR promoter can be used as a candidate promoter for SMA gene therapy drug design.

(2) In vivo evaluation experiment

On the basis of in vitro evaluation experiments, 5 promoter-regulated Fluc gene expression cassettes are further packaged into AAV9 viruses to obtain 5 viruses such as AAV9-CA-Fluc, AAV9-CAS-Fluc, AAV9-CAT-Fluc, AAV9-CAR-Fluc, AAV9-CAP-Fluc and the like. The virus was prepared as described in example 2. After virus preparation, 5 viruses were administered at 1X 10¹²The dose of vg (viral genome)/v was injected via tail vein into the C57BL/6J mouse (purchased from Beijing Huafukang Biotech GmbH), and 5 mice were injected for each virus. After 4 weeks of virus injection, mice were sacrificed, liver and brain tissues were separated, total tissue proteins were extracted using a total tissue cell protein extraction kit (Beijing prilley Gene technology Co., Ltd.), and the protein concentration of the extracted total proteins was measured using a protein quantification kit (BCA method) (Beijing prilley Gene technology Co., Ltd.). According to the determinationConcentration, total protein was diluted to 0.5. mu.g/. mu.L, 20. mu.L of the protein solution was used to measure the level of Fluc expression in cells using the Bright-Glo luciferase assay System (Promega, USA), and the measurement results were expressed in relative light intensity units (RLU). The procedure for the detection was as described in the Bright-Glo luciferase detection System. The levels of Fluc expression in liver and brain tissue for the 5 viruses are shown in fig. 21 (liver) and fig. 22 (brain), respectively. From the results in fig. 21, it can be seen that after 5 viruses transduce mouse liver, the expression level of Fluc of rAAV9-CAR-Fluc virus was the highest, and was significantly higher than that of the other 4 viruses. The expression level of Fluc of the rest 4 viruses in the liver of the mouse is AAV9-CAT-Fluc from high to low>AAV9-CA-Fluc>AAV9-CAS-Fluc>AAV 9-CAP-Fluc. From the results of fig. 22, it can be seen that like in the liver, the level of Fluc expression of rAAV9-CAR-Fluc virus remained the highest in brain tissue and was significantly higher than the other 4 viruses. The expression level sequence of the other 4 viruses is different from the expression level sequence of the viruses in liver, and the expression level sequence of the 4 viruses in brain tissue is specifically AAV9-CA-Fluc>AAV9-CAT-Fluc>AAV9-CAS-Fluc> AAV9-CAP-Fluc。

In vivo evaluation experiment results show that the expression level of the CAR promoter in mouse liver and brain tissues is obviously higher than that of other 4 promoters, and the CAR promoter can be used as a candidate promoter for SMA gene drug design.

According to the results of in-vitro evaluation experiments of the synthesis body, the CAR promoter shows higher expression activity than other 4 promoters in-vivo evaluation experiments, and a new promoter selection is provided for SMA gene drug design. Because the promoter in the AVXS-101 medicine structure is similar to the CA promoter, the SMN1 gene expression box regulated by the CA promoter is used as an available positive control in the subsequent SMA medicine effectiveness evaluation experiment.

Example 4 in vivo efficacy evaluation experiment of SMA Gene drug

In the process of designing SMA gene medicine, we mainly consider 3 factors of optimizing SMN1 gene coding sequence, adding human miR-122 target sequence in gene expression box, and mutating AAV vector Inverted Terminal Repeat (ITR) and the like to SMAThe effect on the in vivo efficacy and safety of gene drugs. Selection of SMN2^+/+, SMNΔ7^+/+, smn^-/-Mice were evaluated for the in vivo efficacy of SMA gene drugs. SMN2^+/+, SMNΔ7^+/+, smn^-/-Mice were prepared with SMN2^+/+, SMNΔ7^+/+, smn^+/-Mice were bred. SMN2^+/+, SMNΔ7^+/+, smn^+/-Seed mice were purchased from the Jackson laboratory, usa, and mouse number 005025. Propagation and genotyping of the models is described in reference to the Jackson laboratory.

Optimization of SMN1 gene coding sequence on SMA gene medicine effectiveness

First, we evaluated the effect of the optimization of the SMN1 gene coding sequence on the drug efficacy of SMA genes. 6 viruses such as AAV9-CA-Fluc, AAV9-CAR-Fluc, scAAV9-CA-SMN1, scAAV9-CAR-SMN1, scAAV9-CA-COSMN1 and scAAV9-CAR-COSMN1 were prepared. The AAV9-CA-Fluc and AAV9-CAR-Fluc are single-chain AAV viruses, are Fluc gene expression cassettes regulated by a CA promoter and a CAR promoter respectively, and are used as negative controls for effectiveness evaluation. 4 viruses such as scAAV9-CA-SMN1, scAAV9-CAR-SMN1, scAAV9-CA-cosMN1, scAAV9-CAR-cosMN1 and the like are double-stranded AAV viruses, and are respectively an SMN1 gene expression frame or an optimized cosMN1 gene expression frame regulated by a CA promoter and a CAR promoter, and are used for comparing the influence of sequence optimization on the drug effectiveness of SMA genes.

6 different recombinant AAV viruses (AAV 9-CA-Fluc, AAV9-CAR-Fluc, scAAV9-CA-SMN1, scAAV9-CA-cosMN1, scAAV9-CAR-SMN1, scAAV9-CAR-cosMN 1) were tested at 5X 10¹³Dose of vg/kg A1 day old model mouse of SMA (SMN 2) was injected via tail vein^+/+, SMNΔ7^+/+, smn^-/-Mice), in which AAV9-CA-Fluc and AAV9-CAR-Fluc were control viruses, 5 SMA model mice were injected for each virus. After virus injection, the survival of the mice was recorded. The results are shown in FIG. 23. From the results in FIG. 23, it can be seen that the survival of mice injected with AAV9-CA-Fluc and AAV9-CAR-Fluc did not exceed 15 days, which is consistent with the literature reports (Le TT, et al Hum Mol Genet 2005; 14(6):845 and 857.). And injecting scAAV9-CA-SMN1 and scAAV9-The survival of SMA model mice with CA-coSMN1, scAAV9-CAR-SMN1 and scAAV9-CAR-coSMN1 virus was prolonged, and the survival of mice injected with model mice containing the CAR promoter virus (scAAV 9-CAR-SMN1 and scAAV9-CAR-coSMN 1) was longer than that of mice injected with model mice containing CA promoter virus (scAAV 9-CA-SMN1 and scAAV9-CA-coSMN 1), and that of mice injected with optimized model mice containing the coSMN1 gene virus (scAAV 9-CA-coSMN1 or scAAV9-CAR-coSMN 1) was longer than that of the respective virus carrying the unoptimized SMN1 gene (scAAV 9-CA-SMN1 or scAAV 9-SMN-CAR 1). The result shows that the CAR promoter element and the SMN1 gene sequence optimization are both helpful to improve the treatment effect of SMA gene treatment drugs on SMA animal models. Furthermore, from the results in fig. 23, we also found that SMA model mice injected with scAAV9-CA-SMN1, scAAV9-CA-coSMN1, scAAV9-CAR-SMN1, scAAV9-CAR-coSMN1 virus all survived shorter than wild-type C57BL/6J mice, but the differences were not large, indicating that the gene drug can significantly prolong the survival of SMA model mice.

Meanwhile, the body weight change of mice injected with SMA models carrying recombinant AAV viruses (AAV 9-CA-Fluc, AAV9-CAR-Fluc, scAAV9-CA-SMN1, scAAV9-CA-COSMN1, scAAV9-CAR-SMN1 and scAAV9-CAR-COSMN 1) with SMN1 or COSMN1 gene expression cassettes was recorded at different time points. In the weight recording process, considering that experimental mice that may have died at the recording time point have been recorded, we record only the weight of the surviving mice, and thus the average value of the weights of the experimental mice at each time point shown in the graph is the average value of the weights of the surviving mice. The results are shown in FIG. 24. From the results in fig. 24, it can be seen that the body weight gain speed of mice injected with AAV9-CA-Fluc and AAV9-CAR-Fluc is significantly lower than that of mice injected with recombinant AAV viruses carrying SMN1 or coSMN1 gene expression cassettes (AAV 9-CA-Fluc, AAV9-CAR-Fluc, scAAV9-CA-SMN1, scAAV9-CA-coSMN1, scAAV9-CAR-SMN1, scAAV9-CAR-coSMN 1) and wild-type mice, and then death and no body weight recording indicates that the mice in SMA model express Fluc gene can not restore its physiological function. The weight increasing speed of the SMA model mice injected with the viruses scAAV9-CA-SMN1, scAAV9-CA-COSMN1, scAAV9-CAR-SMN1 and scAAV9-CAR-COSMN1 is consistent with that of wild type mice, and no obvious difference is found among 4 viruses, so that the growth and development of the SMA model mice can be effectively recovered and the weight is increased after the viruses expressing the SMN1 protein are injected.

Influence of (II) miR-122 target sequence on effectiveness of SMA gene medicament

Next, we compared the effect of the miR-122 target sequence on the drug effectiveness of the SMA gene. 4 viruses such as scAAV9-CAR-SMN1, scAAV9-CAR-cosMN1, scAAV9-CAR-SMN1-122T and scAAV9-CAR-cosMN1-122T are prepared. Wherein the scAAV9-CAR-SMN1 and the scAAV9-CAR-cosMN1 respectively carry an SMN1 gene expression cassette and a cosMN1 gene expression cassette which are regulated by the CAR promoter, and do not contain a human miR-122 target sequence. The scAAV9-CAR-SMN1-122T, scAAV9-CAR-coSMN1-122T respectively carry an SMN1 gene expression cassette and a coSMN1 gene expression cassette which are regulated by a CAR promoter and contain a human miR-122 target sequence, and the expression cassette contains the human miR-122 target sequence.

4 different recombinant AAV viruses (scAAV 9-CAR-SMN1, scAAV9-CAR-cosMN1, scAAV9-CAR-SMN1-122T and scAAV9-CAR-cosMN 1-122T) were tested at 5X 10¹³Dose of vg/kg A1 day old model mouse of SMA (SMN 2) was injected via tail vein^+/+, SMNΔ7^+/+, smn^-/-Mice), 5 SMA model mice were injected with each virus. After virus injection, the survival of the mice was recorded. The results are shown in FIG. 25. From the results in FIG. 25, it can be seen that the duration of survival of mice in SMA model injected with scAAV9-CAR-SMN1-122T and scAAV9-CAR-cosMN1-122T viruses was longer than that of mice in SMA model injected with scAAV9-CAR-SMN1 and scAAV9-CAR-cosMN1 viruses without human miR-122 target sequence, indicating that the addition of human miR-122 target sequence contributes to the improvement of the efficacy of SMA gene therapy drugs in SMA model mice. Furthermore, from the results in fig. 25, we also found that SMA model mice injected with scAAV9-CAR-SMN1, scAAV9-CAR-coSMN1, scAAV9-CAR-SMN1-122T and scAAV9-CAR-coSMN1-122T viruses all survived shorter than wild-type C57BL/6J mice, but the differences were not great, indicating that 4 gene drugs can significantly prolong survival of SMA model mice.

At the same time, we recorded changes in body weight in SMA model mice injected with 4 SMA gene therapy drugs (scAAV 9-CAR-SMN1, scAAV9-CAR-coSMN1, scAAV9-CAR-SMN1-122T and scAAV9-CAR-coSMN 1-122T) at different time points. In the weight recording process, considering that experimental mice that may have died at the recording time point have been recorded, we record only the weight of the surviving mice, and thus the average value of the weights of the experimental mice at each time point shown in the graph is the average value of the weights of the surviving mice. The results are shown in FIG. 26. From the results in fig. 26, it can be seen that the body weight gain rate of SMA model mice injected with scAAV9-CAR-SMN1, scAAV9-CAR-coSMN1, scAAV9-CAR-SMN1-122T and scAAV9-CAR-coSMN1-122T viruses was identical to that of wild type mice, and no significant difference was observed between 4 viruses, indicating that the mice in SMA model could be effectively restored to growth and development and body weight gain after the injection of viruses expressing SMN1 protein.

We further examined the effect of adding the miR-122 target sequence in the expression cassette of the cosMN1 gene on the expression of the cosMN1 gene. 3 months after virus injection, 1 mouse was sacrificed for each of scAAV9-CAR-coSMN1 and scAAV9-CAR-coSMN1-122T virus injection groups, organs such as heart, liver, skeletal muscle, spleen, lung, brain and kidney were isolated, total RNA of each organ was extracted, copy number of coSMN1 RNA and mouse GAPDH RNA (glyceraldehyde-3-phosphate dehydrogenase RNA) in total RNA was determined by quantitative PCR, copy number was expressed as Ct value, difference between Ct value of coSMN1 RNA and Ct value of mouse GAPDH RNA was calculated, and power of difference between Ct values for 2 was expressed relative expression level of coSMN1 RNA. In the detection, an SMA model mouse (SMN 2) without virus injection is used^+/+, SMNΔ7^+/+, smn^-/-) As a control. No changes in SMN1 gene expression were detected in the present invention, the primary consideration being the SMA model mouse (SMN 2)^+/+, SMNΔ7^+/+, smn^-/-) The gene containing human SMN2 (Monani UR, et al, Hum Mol Genet 2000; 9(3): 333-. Therefore, the expression level of SMN1 gene before and after viral transduction was not detected in the present invention.

Quantitative PCR method was used to determine Ct values for SMN1 RNA or coSMN1 RNA and mouse GAPDH RNA in total RNA. The specific process is as follows:

primers and probes for quantitative PCR detection were designed for coSMN1 RNA sequence:

coSMN1-Q-F：5’- CACCACCTCCAATCTGTCCT-3’ (SEQ ID NO.21)

coSMN1-Q-R：5’- TAGTAGCCGGTGTGATAGCC-3’ (SEQ ID NO.22)

coSMN1-Q-P：5’- ACGATGCCGATGCCCTGGGC-3’ (SEQ ID NO.23)。

primers and probes for quantitative PCR detection were designed for the mouse GAPDH RNA sequence:

GAPDH-Q-F：5’-AACGGATTTGGCCGTATTGG-3’ (SEQ ID NO.24)

GAPDH-Q-R：5’-AATCTCCACTTTGCCACTGC-3’ (SEQ ID NO.25)

GAPDH-Q-P：5’-CGCCTGGTCACCAGGGCTGC-3’ (SEQ ID NO.26)

cosMN1-Q-F/cosMN1-Q-R and GAPDH-Q-F/GAPDH-Q-R are primers, and cosMN1-Q-P and GAPDH-Q-P are probes. The 5 'end of the probe is marked by FAM fluorescent protein, and the 3' end is connected with BlackBerry query. Primers and probes were synthesized by thermolfisher Scientific. Specifically amplifying a 97bp fragment in the SMN1 sequence by using cosMN1-Q-F and cosMN1-Q-R as primers, specifically amplifying a 66bp fragment in the GAPDH sequence by using GAPDH-Q-F and GAPDH-Q-R as primers, measuring the amplification Ct values (representing copy numbers) of the SMN1 RNA, cosMN1 RNA and GAPDH RNA in a detection sample by using a One-Step reaction TaqMan probe binding method, applying One Step PrimeScript RT-PCR Kit (Perfect Real Time) reagent (Takara, Dalian, China) and detecting by using a fluorescence quantitative PCR instrument (model: ABI 7500 fast, ABI). The procedure is described in the One Step PrimerScript RT-PCR Kit (Perfect Real Time) reagent instruction.

The results of the quantitative PCR assay are shown in FIG. 27. From the results in FIG. 27, it can be seen that the SMA model mouse (SMN 2) was compared with that of the mouse without virus injection^+/+, SMNΔ7^+/+, smn^-/-) The mice injected with the scAAV9-CAR-cosMN1 and scAAV9-CAR-cosMN1-122T virus group have the advantage that the expression level of the cosMN1 is increased in organs such as cardiac muscle, skeletal muscle, spleen, kidney, brain, lung and the like, which indicates that 2 viruses can be injected intravenously to obtain the miceEfficiently transduces systemic tissue organ expression to produce a coSMN1 RNA molecule. Furthermore, AAV vectors transduce liver efficiently when injected intravenously. The experimental result shows that the target sequence of miR-122 highly expressed in liver added into 3' UTR of the expression cassette of the cosMN1 gene can effectively inhibit the expression of the cosMN1 gene (figure 27), reduce the side effect possibly brought by over-expression of SMN1 protein in liver and increase the effectiveness of gene drugs. The results in FIG. 27 are also shown in SMA model mice (SMN 2)^+/+, SMNΔ7^+/+, smn^-/-) The expression of the coSMN1 gene was not detected because the coSMN1 gene is the coding sequence of the codon-optimized human SMN1 gene and the DNA sequence thereof is different from the human SMN1 RNA sequence generated by SMA model mouse expression, so the probes and primers for quantitative PCR detection of the virus-carried human coSMN1 gene cannot recognize and bind to the human SMN1 RNA sequence generated by SMA model mouse expression, and there is no detection signal.

(III) Effect of AAV vector Inverted Terminal Repeat (ITR) mutation on drug effectiveness of SMA Gene

Finally, we compared the effect of AAV vector Inverted Terminal Repeat (ITR) mutations on SMA gene drug efficacy. 4 viruses such as scAAV9-CAR-coSMN1, scAAV9-CAR-coSMN1-122T, scAAV9-U-CAR-coSMN1 and scAAV9-U-CAR-coSMN1-122T were prepared. Wherein the scAAV9-CAR-cosMN1, scAAV9-CAR-cosMN1-122T respectively carry a cosMN1 gene expression cassette regulated by a CAR promoter and a cosMN1 gene expression cassette containing a miR-122 target sequence, and ITR sequences in AAV virus are a normal ITR sequence and a delta ITR sequence of a deletion D sequence. And the scAAV9-U-CAR-cosMN1 and scAAV9-U-CAR-cosMN1-122T respectively carry a cosMN1 gene expression cassette regulated by a CAR promoter and a cosMN1 gene expression cassette containing a miR-122 target sequence, but AAV virus ITR sequences are a U-ITR sequence deleting a B-B 'sequence and a C-C' sequence and a delta U-ITR sequence deleting a B-B 'sequence, a C-C' sequence and a D sequence.

4 different recombinant AAV viruses (scAAV 9-C)AR-cosMN1, scAAV9-CAR-cosMN1-122T, scAAV9-U-CAR-cosMN1 and scAAV9-U-CAR-cosMN 1-122T) at 5X 10¹³Dose of vg/kg A1 day old model mouse of SMA (SMN 2) was injected via tail vein^+/+, SMNΔ7^+/+, smn^-/-Mice), 5 SMA model mice were injected with each virus. After virus injection, the survival of the mice was recorded. The results are shown in FIG. 28. From the results in FIG. 28, it can be seen that the mice injected with the SMA model of the scAAV9-U-CAR-coSMN1 and scAAV9-U-CAR-coSMN1-122T viruses had longer survival than the mice injected with the SMA model of the scAAV9-CAR-coSMN1, scAAV9-CAR-coSMN1-122T viruses, indicating that the deletion of the U-ITR sequences derived from the B-B 'and C-C' sequences in the AAV ITR sequences contributed to the improvement in the efficacy of the SMA gene therapy drugs in the mice of the SMA model. Furthermore, from the results in fig. 28, we also know that SMA model mice injected with scAAV9-CAR-coSMN1, scAAV9-CAR-coSMN1-122T, scAAV9-U-CAR-coSMN1, and scAAV9-U-CAR-coSMN1-122T virus all survived shorter than wild-type C57BL/6J mice, but the differences were not great, indicating that 4 gene drugs can significantly prolong the survival of SMA model mice.

At the same time, we recorded the body weight changes of SMA model mice injected with 4 SMA gene therapy drugs (scAAV 9-CAR-coSMN1, scAAV9-CAR-coSMN1-122T, scAAV9-U-CAR-coSMN1 and scAAV9-U-CAR-coSMN 1-122T) at different time points. In the weight recording process, considering that experimental mice that may have died at the recording time point have been recorded, we record only the weight of the surviving mice, and thus the average value of the weights of the experimental mice at each time point shown in the graph is the average value of the weights of the surviving mice. The results are shown in FIG. 29. From the results in fig. 29, it can be seen that the body weight gain rate of SMA model mice injected with scAAV9-CAR-coSMN1, scAAV9-CAR-coSMN1-122T, scAAV9-U-CAR-coSMN1 and scAAV9-U-CAR-coSMN1-122T viruses was identical to that of wild type mice, and no significant difference was observed between 4 viruses, indicating that the mice expressing SMN1 protein could effectively recover their growth and development and gain weight.

We further tested 4 SMA gene therapy drugs injected (scAAV 9-CAR-cosMN1, scAAV9-CAR-cosMN1-122T, scAAV9-U-CAR-cosMN1 andscAAV9-U-CAR-coSMN 1-122T), coSMN1 gene expression in different tissues of SMA model mice. After 3 months of virus injection, 1 mouse was sacrificed each group, organs such as heart, liver, skeletal muscle, spleen, lung, brain and kidney were isolated, total RNA of each organ was extracted, copy number of coSMN1 RNA and copy number of mouse GAPDH RNA in total RNA were determined by quantitative PCR, copy number was expressed by Ct value, difference between Ct value of coSMN1 RNA and Ct value of mouse GAPDH RNA was calculated, and power of difference for Ct value of 2 represents relative expression level of coSMN1 RNA. In the detection, an SMA model mouse (SMN 2) without virus injection is used^+/+, SMNΔ7^+/+, smn^-/-) As a control.

Quantitative PCR method was used to determine Ct values for cosMN1 RNA and mouse GAPDH RNA in total RNA. The specific process is as follows:

primers and probes for quantitative PCR detection were designed for coSMN1 RNA sequence:

coSMN1-Q-F：5’- CACCACCTCCAATCTGTCCT-3’ (SEQ ID NO.21)

coSMN1-Q-R：5’- TAGTAGCCGGTGTGATAGCC-3’ (SEQ ID NO.22)

coSMN1-Q-P：5’- ACGATGCCGATGCCCTGGGC-3’ (SEQ ID NO.23)。

primers and probes for quantitative PCR detection were designed for the mouse GAPDH RNA sequence:

GAPDH-Q-F：5’-AACGGATTTGGCCGTATTGG-3’ (SEQ ID NO.24)

GAPDH-Q-R：5’-AATCTCCACTTTGCCACTGC-3’ (SEQ ID NO.25)

GAPDH-Q-P：5’-CGCCTGGTCACCAGGGCTGC-3’ (SEQ ID NO.26)

cosMN1-Q-F/cosMN1-Q-R and GAPDH-Q-F/GAPDH-Q-R are primers, and cosMN1-Q-P and GAPDH-Q-P are probes. The 5 'end of the probe is marked by FAM fluorescent protein, and the 3' end is connected with BlackBerry query. Primers and probes were synthesized by thermolfisher Scientific. Specifically amplifying a 97bp fragment in the SMN1 sequence by using cosMN1-Q-F and cosMN1-Q-R as primers, specifically amplifying a 66bp fragment in the GAPDH sequence by using GAPDH-Q-F and GAPDH-Q-R as primers, measuring the amplification Ct values (representing copy numbers) of the SMN1 RNA, cosMN1 RNA and GAPDH RNA in a detection sample by using a One-Step reaction TaqMan probe binding method, applying One Step PrimeScript RT-PCR Kit (Perfect Real Time) reagent (Takara, Dalian, China) and detecting by using a fluorescence quantitative PCR instrument (model: ABI 7500 fast, ABI). The procedure is described in the One Step PrimerScript RT-PCR Kit (Perfect Real Time) reagent instruction.

The results of the quantitative PCR assay are shown in FIG. 30. From the results in FIG. 30, it can be seen that the SMA was compared with that of the mouse model (SMN 2) in which no virus was injected^+/+, SMNΔ7^+/+, smn^-/-) In mice of viral groups of scAAV9-CAR-cosMN1, scAAV9-CAR-cosMN1-122T, scAAV9-U-CAR-cosMN1 and scAAV9-U-CAR-cosMN1-122T, the expression level of cosMN1 in organs such as cardiac muscle, skeletal muscle, spleen, kidney, brain and lung is increased, which indicates that 4 viruses can effectively transduce the expression of tissues and organs of the whole body to generate SMN1 RNA or cosMN1 RNA molecules after intravenous injection. In addition, in the brain, the expression level of the cosMN1 of the mice injected with the scAAV9-U-CAR-cosMN1 and scAAV9-U-CAR-cosMN1-122T virus group is obviously higher than that of the mice injected with the scAAV9-CAR-cosMN1 and scAAV9-CAR-cosMN1-122T virus group, and U-ITR can improve the gene expression level after AAV virus transduction in vivo and is helpful for improving the effectiveness of SMA gene drug design. Furthermore, our experimental results also show that the expression level of the coSMN1 gene in the liver of mice injected with miR-122 target viruses (scAAV 9-CAR-coSMN1-122T and scAAV9-U-CAR-coSMN 1-122T) is significantly lower than that of mice injected with viruses not containing miR-122 target (scAAV 9-CAR-coSMN1 and scAAV9-U-CAR-coSMN 1) (fig. 30), indicating that the addition of the 3' UTR of the expression cassette of coSMN1 gene to the liver highly expressed miR-122 target sequence effectively inhibits the expression of the coSMN1 gene in the liver, reduces the side effects that the overexpression of SMN1 protein in the liver may bring, and increases the effectiveness of gene drugs.

In conclusion, in this example, we analyzed the influence of 3 factors, such as whether the SMN1 gene coding sequence was optimized, whether the human miR-122 target sequence was added to the gene expression cassette, whether the AAV vector inverted terminal repeat sequence (ITR) was mutated, etc., on the in vivo effectiveness and safety of SMA gene drugs through experimental comparison, and found that the optimization of the SMN1 gene coding sequence, the addition of the human miR-122 target sequence to the expression cassette, and the AAV vector inverted terminal repeat sequence (ITR) all contribute to improving the in vivo effectiveness of SMA gene drugs. Therefore, the SMA drug structure can be designed to simultaneously consider 3 factors or 1 factor of the 3 factors and 2 factor combinations of the 3 factors.

Example 5 in vivo efficacy of SMA Gene drugs by different modes of administration

On the basis of verifying the effectiveness of the designed medicament, the influence of different administration modes on the in-vivo effectiveness of the designed SMA gene medicament is further compared. Two viruses, namely scAAV9-CAR-cosMN1 and scAAV9-CAR-cosMN1-122T, are prepared and injected into an SMA model mouse (SMN 2) in 3 different modes of administration, such as intravenous Injection (IV), intrathecal Injection (IT) and intracerebroventricular Injection (ICV) respectively^+/+, SMNΔ7^+/+, smn^-/-Mouse), survival, weight change and coSMN1 expression levels of SMA model mice were compared between different injection regimens.

ScAAV9-CAR-cosMN1 or scAAV9-CAR-cosMN1-122T were injected into 1-day-old SMA model mice (SMN 2) by 3 different administration methods, i.e., intravenous Injection (IV), intrathecal Injection (IT), and intracerebroventricular Injection (ICV)^+/+, SMNΔ7^+/+, smn^-/-Mice) 5 mice were injected with each virus by one injection. Intravenous (IV) administration was 5X 10¹³vg/kg, intrathecal Injection (IT) and intracerebroventricular Injection (ICV) were all administered at a dose of 1X 10¹³vg/kg. After injection was complete, the survival of the mice was recorded. The results are shown in FIG. 31. As can be seen from the results in fig. 31, although the injection dose for intrathecal injection and intracerebroventricular injection was 5-fold lower than the injection dose for intravenous injection, no significant difference was observed in the duration of survival extension between the 3 injection-type mice. Moreover, in the 3 injection modes, the survival time of the mice injected with the miR-122 target sequence virus is prolonged than that of the mice injected with the miR-122 target sequence virus, which is consistent with the previous findings.

At the same time, we also recorded the body weight of the virus-injected mice at different time points, and the results are shown in fig. 32. From the results in FIG. 32, it is clear that the body weight of the mice injected with 3 types of injection gradually increased with time until the body weight was unchanged, which is consistent with the body weight change rule of the wild-type C57BL/6J mice, indicating that the 3 types of injection can effectively recover the growth and development of the SMA model mice.

We further examined the expression of the coSMN1 gene in different tissues of the SMA model mice after 3 different injection modalities (i.v., intrathecal injection, and intracerebroventricular injection) of SMA gene drugs (either scAAV9-CAR-coSMN1 or scAAV9-CAR-coSMN 1-122T). After 3 months of virus injection, 1 mouse was sacrificed each group, organs such as heart, liver, skeletal muscle, spleen, lung, brain and kidney were isolated, total RNA of each organ was extracted, copy number of coSMN1 RNA and copy number of mouse GAPDH RNA in total RNA were determined by quantitative PCR, copy number was expressed by Ct value, difference between Ct value of coSMN1 RNA and Ct value of mouse GAPDH RNA was calculated, and power of difference for Ct value of 2 represents relative expression level of coSMN1 RNA. In the detection, an SMA model mouse (SMN 2) without virus injection is used^+/+, SMNΔ7^+/+, smn^-/-) As a control. The specific detection process is detailed in example 5. The detection results are shown in fig. 33. From the results in fig. 33, it can be seen that the expression level of coSMN1 was significantly increased in the brains of SMA model mice administered by intrathecal injection and intraventricular injection compared to SMA model mice administered by intravenous injection, indicating that intrathecal injection and intraventricular injection are more likely to transduce neurons than intravenous injection. Furthermore, we found that coSMN1 expression was also detected in tissues such as liver, skeletal muscle, and cardiac muscle of mice administered with intrathecal injection and intracerebroventricular injection. Although the expression level of coSMN1 gene in tissues such as liver, skeletal muscle and cardiac muscle of mice was lower than that of intravenous injection in these two administration modes, the difference was not great and was consistent with the literature report (Armbruster N, et al Mol Ther Methods Clin dev. 2016;3: 16060.), suggesting that AAV vector penetration through the blood brain barrier may be bidirectional. Our experimental results also show that the expression level of the coSMN1 gene in the liver of mice with miR-122 target virus (scAAV 9-CAR-coSMN 1-122T) was significantly lower for all 3 injections than for mice without miR-122 target virus (scAAV 9-CAR-coSMN1 and scAAV9-U-CAR-coSMN 1) (fig. 33), indicating that the addition of the 3' UTR of the expression cassette of the coSMN1 gene to the liver of highly expressed miR-122 target (miR-122) was shownEffectively inhibits the expression of the cosMN1 gene in the liver, reduces the possible side effect caused by over-expression of the SMN1 protein in the liver, and increases the effectiveness of gene drugs.

In summary, in this example, we comparatively analyzed the in vivo efficacy impact of 3 different modes of administration (i.v., intrathecal injection and intracerebroventricular injection) on the design of SMA gene drugs. The results show that the 3 administration modes can effectively prolong the survival time of the SMA model mouse and increase the weight of the SMA model mouse, and the effective dose required by intrathecal injection and intracerebroventricular injection is lower, thereby providing a new choice for the administration mode of the SMA gene medicament.

Example 6 in vivo efficacy of different AAV vector serotypes on SMA Gene drugs

In this example, we compared the effect of different AAV vector serotypes on the in vivo efficacy of drug design of SMA genes. We prepared 3 viruses such as scAAV5-CAR-cosMN1, scAAV9-CAR-cosMN1, scAAVrh10-CAR-cosMN1, and injected the 3 viruses into SMA model mice (SMN 2) by 3 different administration modes such as intravenous Injection (IV), intrathecal Injection (IT) and intracerebroventricular Injection (ICV) respectively^+/+, SMNΔ7^+/+, smn^-/-Mice), the survival time and the weight change of mice injected with different AAV serotype viruses SMA models under the same injection mode are compared.

(one) Effect of AAV vector serotypes on in vivo efficacy of SMA Gene Agents upon intravenous administration

The scAAV5-CAR-cosMN1, scAAV9-CAR-cosMN1 or scAAVrh10-CAR-cosMN1 virus was intravenously administered at 5X 10¹³Dose of vg/kg was injected into 1 day old SMA model mice (SMN 2)^+/+, SMNΔ7^+/+, smn^-/-Mice) in vivo, 5 mice were injected for each virus. After injection was complete, the survival of the mice was recorded. The results are shown in FIG. 34. From the results in FIG. 34, it can be seen that 3 viruses can prolong survival of mice in SMA model after injection, but different AAV vector serotypes have different degrees of prolongation of survival of mice in SMA model, and AAV9(scAAV 9-CAR-COSMN 1) has the best effect, AAVrh10 (scAAVrh 10-CAR-COSMN 1) has the second best effect, and AAV5 (scAAV 5-CAR-COSMN 1) has the best effectWeak. Nevertheless, the mean survival of SMA model mice injected with scAAV5-CAR-coSMN1 virus was 285.5 days, significantly longer than the expected survival of SMA model mice (no more than 15 days), and therefore both AAVrh10 and AAV9, including AAV5, could be candidate serotypes for SMA gene drug design.

At the same time, we also recorded the body weight of the virus-injected mice at different time points, and the results are shown in fig. 35. From the results in fig. 35, it can be seen that the body weight of the mice gradually increased with time after the injection of different serotypes of AAV viruses until the body weight is not changed, which is consistent with the body weight change rule of wild type C57BL/6J mice, and it is demonstrated that 3 serotypes of AAV viruses (scAAV 5-CAR-coSMN1, scAAV9-CAR-coSMN1 and scAAVrh10-CAR-coSMN 1) can effectively restore the growth and development of SMA model mice.

(II) Effect of AAV vector serotypes on in vivo efficacy of SMA Gene drugs upon intrathecal administration

Intrathecal 1X 10 of scAAV5-CAR-cosMN1, scAAV9-CAR-cosMN1 or scAAVrh10-CAR-cosMN1 viruses¹³Dose of vg/kg was injected into 1 day old SMA model mice (SMN 2)^+/+, SMNΔ7^+/+, smn^-/-Mice) in vivo, 5 mice were injected for each virus. After injection was complete, the survival of the mice was recorded. The results are shown in FIG. 36. From the results in fig. 36, it can be seen that the survival of SMA model mice was prolonged by viral injection of scAAV5-CAR-coSMN1, scAAV9-CAR-coSMN1, or scAAV vrh10-CAR-coSMN1, but the survival of SMA model mice was different for different AAV vector serotypes, and AAV9(scAAV 9-CAR-coSMN 1) was most effective, AAVrh10 (scAAV 10-CAR-coSMN 1) was less effective, and AAV5 (scAAV 5-CAR-coSMN 1) was least effective. The mean survival time of SMA model mice injected with scAAV5-CAR-cosMN1 virus is 299 days, which is significantly longer than the expected survival time of SMA model mice (no more than 15 days), so AAVrh10 and AAV9 including AAV5 can be used as candidate serotypes of SMA gene drug design.

At the same time, we also recorded the body weight of the virus-injected mice at different time points, and the results are shown in fig. 37. From the results in fig. 37, it can be seen that the body weight of the mice gradually increased with time after the injection of different serotypes of AAV viruses until the mice are not changed, which is consistent with the body weight change rule of wild type C57BL/6J mice, and it is demonstrated that the growth and development of the SMA model mice can be effectively recovered after the intrathecal injection of 3 serotypes of AAV viruses (scAAV 5-CAR-coSMN1, scAAV9-CAR-coSMN1 and scAAVrh10-CAR-coSMN 1).

(III) Effect of AAV vector serotypes on in vivo efficacy of SMA Gene drugs upon intracerebroventricular injection administration

The scAAV5-CAR-cosMN1, scAAV9-CAR-cosMN1 or scAAVrh10-CAR-cosMN1 viruses were intracerebroventricularly at 1 × 10¹³Dose of vg/kg was injected into 1 day old SMA model mice (SMN 2)^+/+, SMNΔ7^+/+, smn^-/-Mice) in vivo, 5 mice were injected for each virus. After injection was complete, the survival of the mice was recorded. The results are shown in FIG. 38. From the results in fig. 38, 3 viruses were able to prolong survival after SMA model mice injection, but the different AAV vector serotypes differed in their prolongation of survival in SMA model mice, with AAV9(scAAV 9-CAR-coSMN 1) and AAVrh10 (scAAVrh 10-CAR-coSMN 1) being equally effective, and AAV5 (scAAV 5-CAR-coSMN 1) being the weakest. Nevertheless, the mean survival of SMA model mice injected with scAAV5-CAR-coSMN1 virus was 304 days, significantly longer than the expected survival of SMA model mice (no more than 15 days), and therefore both AAVrh10 and AAV9, including AAV5, were candidate serotypes of SMA gene drug design.

At the same time, we also recorded the body weight of the virus-injected mice at different time points, and the results are shown in fig. 39. From the results in fig. 39, it can be seen that the body weight of the mice gradually increased with time after the injection of different serotypes of AAV viruses until the body weight is not changed, which is consistent with the body weight change rule of wild type C57BL/6J mice, and it is demonstrated that 3 serotypes of AAV viruses (scAAV 5-CAR-coSMN1, scAAV9-CAR-coSMN1 and scAAVrh10-CAR-coSMN 1) can effectively restore the growth and development of SMA model mice.

In summary, in this example, we compared the effect of 3 serotypes, AAV5, AAV9, and AAVrh10, on the efficacy of SMA gene therapy drugs. Although the effectiveness of different serotypes is different to a certain extent, the SMA gene medicine based on 3 serotypes of AAV can remarkably prolong the survival period of an SMA animal model and recover the physiological function of the model. Moreover, the 3 serotypes show similar rules under 3 different administration modes (intravenous injection, intrathecal injection and intracerebroventricular injection), and the two serotypes of AAV5 and AAVrh10 except AAV9 can be used for designing SMA gene therapy medicines.

Sequence listing

<110> research institute of gene therapy technology for Beijing Raichh's disease

BEIJING FIVEPLUS MOLECULAR MEDICINE INSTITUTE Co.,Ltd.

THE SECOND HOSPITAL OF HEBEI MEDICAL University

<120> recombinant adeno-associated virus carrying artificially designed SMN1 gene expression cassette and application thereof

<160> 26

<170> SIPOSequenceListing 1.0

<210> 1

<211> 660

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg 60

tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 120

gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca 180

gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 240

tacccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc 300

cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg 360

gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc 420

ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga 480

ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcgacg cgctaagcgg 540

ccgcgtggga gggcgataac cactcgtaga aagcgtgaga agttactaca agcggtcctc 600

ccggccaccg tactgttccg ctcccagaag ccccgggcgg cggaagtcgt cactcttaag 660

<210> 2

<211> 628

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg 60

tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 120

gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca 180

gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 240

tacccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc 300

cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg 360

gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc 420

ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga 480

ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcgacg cgctatgcgg 540

ccgcgtgagg cggagggagc gggtgatgtt agaaggcggg cgaggaatgg ctccaggact 600

aacccctggc acctccccct ccccacag 628

<210> 3

<211> 631

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg 60

tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 120

gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca 180

gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 240

tacccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc 300

cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg 360

gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc 420

ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga 480

ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcgacg cgctatgcgg 540

ccgcgtgagt gagtgatttg atctaaatgc ctttttgtaa tcaagtgact gtgtgtgtgt 600

gtatctgagt gcctgattct tttcatcaca g 631

<210> 4

<211> 665

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg 60

tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 120

gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca 180

gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 240

tacccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc 300

cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg 360

gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc 420

ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga 480

ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcgacg cgctaagcgg 540

ccgcgtgtgg ggcggtagtg tgggccctgt tcctgcccgc gcggtgttcc gcattctgca 600

agcctccgga gcgcacgtcg gcagtcggct ccctcgttga ccgaatcacc gacctctctc 660

cccag 665

<210> 5

<211> 527

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg 60

tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 120

gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca 180

gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 240

tacccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc 300

cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg 360

gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc 420

ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga 480

ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcg 527

<210> 6

<211> 121

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ccactccctc tctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120

a 121

<210> 7

<211> 855

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ccactccctc tctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120

aagatcccca ggaagctcct ctgtgtcctc ataaacccta acctcctcta cttgagagga 180

cattccaatc ataggctgcc catccaccct ctgtgtcctc ctgttaatta ggtcacttaa 240

caaaaaggaa attgggtagg ggtttttcac agaccgcttt ctaagggtaa ttttaaaata 300

tctgggaagt cccttccact gctgtgttcc agaagtgttg gtaaacagcc cacaaatgtc 360

aacagcagaa acatacaagc tgtcagcttt gcacaagggc ccaacaccct gctcatcaag 420

aagcactgtg gttgctgtgt tagtaatgtg caaaacagga ggcacatttt ccccacctgt 480

gtaggttcca aaatatctag tgttttcatt tttacttgga tcaggaaccc agcactccac 540

tggataagca ttatccttat ccaaaacagc cttgtggtca gtgttcatct gctgactgtc 600

aactgtagca ttttttgggg ttacagtttg agcaggatat ttggtcctgt agtttgctaa 660

cacaccctgc agctccaaag gttccccacc aacagcaaaa aaatgaaaat ttgacccttg 720

aatgggtttt ccagcaccat tttcatgagt tttttgtgtc cctgaatgca agtttaacat 780

agcagttacc ccaataacct cagttttaac agtaacagct tcccacatca aaatatttcc 840

acaggttaag tcctc 855

<210> 8

<211> 111

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgcggggcg gcctcagtga gcgagcgagc gcgcagagag ggaaaatcca a 111

<210> 9

<211> 845

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgcggggcg gcctcagtga gcgagcgagc gcgcagagag ggaaaatcca aagatcccca 120

ggaagctcct ctgtgtcctc ataaacccta acctcctcta cttgagagga cattccaatc 180

ataggctgcc catccaccct ctgtgtcctc ctgttaatta ggtcacttaa caaaaaggaa 240

attgggtagg ggtttttcac agaccgcttt ctaagggtaa ttttaaaata tctgggaagt 300

cccttccact gctgtgttcc agaagtgttg gtaaacagcc cacaaatgtc aacagcagaa 360

acatacaagc tgtcagcttt gcacaagggc ccaacaccct gctcatcaag aagcactgtg 420

gttgctgtgt tagtaatgtg caaaacagga ggcacatttt ccccacctgt gtaggttcca 480

aaatatctag tgttttcatt tttacttgga tcaggaaccc agcactccac tggataagca 540

ttatccttat ccaaaacagc cttgtggtca gtgttcatct gctgactgtc aactgtagca 600

ttttttgggg ttacagtttg agcaggatat ttggtcctgt agtttgctaa cacaccctgc 660

agctccaaag gttccccacc aacagcaaaa aaatgaaaat ttgacccttg aatgggtttt 720

ccagcaccat tttcatgagt tttttgtgtc cctgaatgca agtttaacat agcagttacc 780

ccaataacct cagttttaac agtaacagct tcccacatca aaatatttcc acaggttaag 840

tcctc 845

<210> 10

<211> 87

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ccactccctc tctgcgcgct cgctcgctca ctgaggccgc ggggcggcct cagtgagcga 60

gcgagcgcgc agagagggaa aatccaa 87

<210> 11

<211> 100

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ccactccctc tctgcgcgct cgctcgctca ctgaggccgc ggggcggcct cagtgagcga 60

gcgagcgcgc agagagggaa aatccaagct agaacaacaa 100

<210> 12

<211> 891

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gccaccatgg cgatgagcag cggcggcagt ggtggcggcg tcccggagca ggaggattcc 60

gtgctgttcc ggcgcggcac aggccagagc gatgattctg acatttggga tgatacagca 120

ctgataaaag catatgataa agctgtggct tcatttaagc atgctctaaa gaatggtgac 180

atttgtgaaa cttcgggtaa accaaaaacc acacctaaaa gaaaacctgc taagaagaat 240

aaaagccaaa agaagaatac tgcagcttcc ttacaacagt ggaaagttgg ggacaaatgt 300

tctgccattt ggtcagaaga cggttgcatt tacccagcta ccattgcttc aattgatttt 360

aagagagaaa cctgtgttgt ggtttacact ggatatggaa atagagagga gcaaaatctg 420

tccgatctac tttccccaat ctgtgaagta gctaataata tagaacaaaa tgctcaagag 480

aatgaaaatg aaagccaagt ttcaacagat gaaagtgaga actccaggtc tcctggaaat 540

aaatcagata acatcaagcc caaatctgct ccatggaact cttttctccc tccaccaccc 600

cccatgccag ggccaagact gggaccagga aagccaggtc taaaattcaa tggcccacca 660

ccgccaccgc caccaccacc accccactta ctatcatgct ggctgcctcc atttccttct 720

ggaccaccaa taattccccc accacctccc atatgtccag attctcttga tgatgctgat 780

gctttgggaa gtatgttaat ttcatggtac atgagtggct atcatactgg ctattatatg 840

ggtttcagac aaaatcaaaa agaaggaagg tgctcacatt ccttaaatta a 891

<210> 13

<211> 891

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gccaccatgg ccatgagctc cggaggatct ggaggaggcg tgcctgagca ggaggacagc 60

gtgctgttcc ggagaggcac cggccagagc gacgattccg acatctggga cgatacagcc 120

ctgatcaagg cctacgataa ggccgtggcc tcctttaagc acgccctgaa gaacggcgat 180

atctgcgaga ccagcggcaa gcctaagacc acaccaaagc ggaagcccgc caagaagaac 240

aagtcccaga agaagaatac agccgcctct ctgcagcagt ggaaagtggg cgacaagtgc 300

tccgccatct ggtctgagga tggctgtatc tatcccgcca ccatcgcctc catcgacttc 360

aagcgggaga cctgcgtggt ggtgtacaca ggctatggca acagagagga gcagaatctg 420

agcgatctgc tgtccccaat ctgtgaggtg gccaacaata tcgagcagaa cgcccaggag 480

aacgagaatg agtctcaggt gagcacagac gagtccgaga acagccggag cccaggaaac 540

aagtctgata atatcaagcc taagtctgcc ccatggaaca gcttcctgcc ccctccaccc 600

cctatgccag gacctaggct gggaccaggc aagcccggcc tgaagtttaa tggacctccc 660

ccacctcctc caccaccacc tccacacctg ctgagctgct ggctgccacc tttcccatcc 720

ggaccaccaa tcatccctcc accacctcca atctgtcctg acagcctgga cgatgccgat 780

gccctgggct ctatgctgat cagctggtac atgtccggct atcacaccgg ctactatatg 840

ggctttaggc agaaccagaa ggagggccgc tgttcccact ctctgaattg a 891

<210> 14

<211> 917

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gccaccatgg cgatgagcag cggcggcagt ggtggcggcg tcccggagca ggaggattcc 60

gtgctgttcc ggcgcggcac aggccagagc gatgattctg acatttggga tgatacagca 120

ctgataaaag catatgataa agctgtggct tcatttaagc atgctctaaa gaatggtgac 180

atttgtgaaa cttcgggtaa accaaaaacc acacctaaaa gaaaacctgc taagaagaat 240

aaaagccaaa agaagaatac tgcagcttcc ttacaacagt ggaaagttgg ggacaaatgt 300

tctgccattt ggtcagaaga cggttgcatt tacccagcta ccattgcttc aattgatttt 360

aagagagaaa cctgtgttgt ggtttacact ggatatggaa atagagagga gcaaaatctg 420

tccgatctac tttccccaat ctgtgaagta gctaataata tagaacaaaa tgctcaagag 480

aatgaaaatg aaagccaagt ttcaacagat gaaagtgaga actccaggtc tcctggaaat 540

aaatcagata acatcaagcc caaatctgct ccatggaact cttttctccc tccaccaccc 600

cccatgccag ggccaagact gggaccagga aagccaggtc taaaattcaa tggcccacca 660

ccgccaccgc caccaccacc accccactta ctatcatgct ggctgcctcc atttccttct 720

ggaccaccaa taattccccc accacctccc atatgtccag attctcttga tgatgctgat 780

gctttgggaa gtatgttaat ttcatggtac atgagtggct atcatactgg ctattatatg 840

ggtttcagac aaaatcaaaa agaaggaagg tgctcacatt ccttaaatta agatccaaac 900

accattgtca cactcca 917

<210> 15

<211> 917

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gccaccatgg ccatgagctc cggaggatct ggaggaggcg tgcctgagca ggaggacagc 60

gtgctgttcc ggagaggcac cggccagagc gacgattccg acatctggga cgatacagcc 120

ctgatcaagg cctacgataa ggccgtggcc tcctttaagc acgccctgaa gaacggcgat 180

atctgcgaga ccagcggcaa gcctaagacc acaccaaagc ggaagcccgc caagaagaac 240

aagtcccaga agaagaatac agccgcctct ctgcagcagt ggaaagtggg cgacaagtgc 300

tccgccatct ggtctgagga tggctgtatc tatcccgcca ccatcgcctc catcgacttc 360

aagcgggaga cctgcgtggt ggtgtacaca ggctatggca acagagagga gcagaatctg 420

agcgatctgc tgtccccaat ctgtgaggtg gccaacaata tcgagcagaa cgcccaggag 480

aacgagaatg agtctcaggt gagcacagac gagtccgaga acagccggag cccaggaaac 540

aagtctgata atatcaagcc taagtctgcc ccatggaaca gcttcctgcc ccctccaccc 600

cctatgccag gacctaggct gggaccaggc aagcccggcc tgaagtttaa tggacctccc 660

ccacctcctc caccaccacc tccacacctg ctgagctgct ggctgccacc tttcccatcc 720

ggaccaccaa tcatccctcc accacctcca atctgtcctg acagcctgga cgatgccgat 780

gccctgggct ctatgctgat cagctggtac atgtccggct atcacaccgg ctactatatg 840

ggctttaggc agaaccagaa ggagggccgc tgttcccact ctctgaattg agatccaaac 900

accattgtca cactcca 917

<210> 16

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ataggtaccg ccaccatgga agatgcc 27

<210> 17

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

attagatctt tacacggcga tcttgcc 27

<210> 18

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

tgggactttc ctacttggca 20

<210> 19

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ggagagtgaa gcagaacgtg 20

<210> 20

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

acccatggtc gaggtgagcc c 21

<210> 21

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

caccacctcc aatctgtcct 20

<210> 22

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

tagtagccgg tgtgatagcc 20

<210> 23

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

acgatgccga tgccctgggc 20

<210> 24

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

aacggatttg gccgtattgg 20

<210> 25

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

aatctccact ttgccactgc 20

<210> 26

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

cgcctggtca ccagggctgc 20

Claims (18)

1. An SMN1 gene expression cassette comprising a CAR promoter represented by SEQ ID NO.3 and a gene encoding human SMN1 protein represented by SEQ ID NO. 13; the SMN1 gene is a human motor neuron survival gene.

2. The SMN1 gene expression cassette of claim 1, wherein the expression cassette further carries a human miRNA target sequence.

3. The SMN1 gene expression cassette of claim 2, wherein the human miRNA target sequence is a target sequence fully complementary to human miR-122.

4. The SMN1 gene expression cassette of claim 3, wherein the number of miRNA target sequences is 1.

5. The SMN1 gene expression cassette of claim 3, wherein the number of miRNA target sequences is greater than 1 and greater than 1 target sequences are connected in series by a spacer sequence.

6. The SMN1 gene expression cassette of claim 3, wherein the SMN1 gene expression cassette comprises the SMN1 coding sequence bearing the miR-122 target sequence shown in SEQ ID NO: 15.

7. The SMN1 gene expression cassette of claim 1, wherein the expression cassette comprises:

(I) a CAR promoter represented by SEQ ID NO. 3; and

(II) the coding sequence of the human SMN1 gene shown in SEQ ID NO: 13; and

(III) human miR-122 target sequence.

8. A recombinant adeno-associated viral vector carrying the SMN1 gene expression cassette of any one of claims 1-7.

9. The recombinant adeno-associated viral vector according to claim 8, wherein the serotype of the recombinant adeno-associated viral vector is selected from the group consisting of: AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh 10.

10. The recombinant adeno-associated viral vector according to claim 9 wherein the serotype is AAV5, AAV9 or AAVrh 10.

11. The recombinant adeno-associated viral vector according to claim 8 wherein the recombinant adeno-associated viral vector comprises a single inverted terminal repeat mutant or 2 identical inverted terminal repeat mutants or 2 different inverted terminal repeat mutants.

12. The recombinant adeno-associated viral vector according to claim 11 wherein the mutant inverted terminal repeats are selected from the group consisting of SEQ ID No.6, SEQ ID No.8 and SEQ ID No. 10.

13. A gene drug comprising the gene expression cassette of any one of claims 1 to 7 or the recombinant adeno-associated virus vector of any one of claims 8 to 12.

14. The gene drug of claim 13, which is an intravenous drug, a intrathecal drug or an intracerebroventricular drug.

15. Use of the recombinant adeno-associated viral vector according to any one of claims 8 to 12 in the manufacture of a medicament for effectively alleviating or curing adverse symptoms caused by a mutation in the human motoneuron survival gene SMN 1.

16. The use of claim 15, wherein the drug is an intravenous drug, an intrathecal drug, or an intracerebroventricular drug.

17. The use of claim 15, wherein the medicament is administered by intravenous injection in combination with intrathecal injection or intracerebroventricular injection.

18. A promoter, the sequence of which is SEQ ID NO. 3.

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