RU2801848C1 - Genetic construct adapted to deliver the human smn1 gene with adeno-associated virus of serotype 2 to provide neurospecific expression - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a genetic construct carrying an open reading frame of the human SMN1 gene, controlled by a neurospecific promoter of the synapsin I (SYN1) gene, and can be used in medicine. Due to the SYN1 promoter contained in the vector, transgene expression will be ensured only in neuronal cells, and the presence of inverted AAV-ITR terminal repeats ensures packaging of the recombinant adeno-associated virus into virions.

EFFECT: invention can be used in the production of a gene therapy medicinal product for combating spinal muscular atrophy type 1 (SMA1) by delivering a construct using a recombinant adeno-associated virus of serotype 2.

1 cl, 1 dwg

Description

Technical field

The invention relates to the fields of molecular biotechnology and medicine and concerns a genetic construct carrying an open reading frame of the human SMN1 gene under the control of a neurospecific synapsin I (SYN1) gene promoter. The invention can serve as the basis for creating a gene therapy drug for combating type I spinal muscular atrophy (SMA1).

The work was financially supported by the Federal State Budgetary Institution "Fund for the Promotion of Small Forms of Enterprises in the Scientific and Technical Sphere" (Fund for the Promotion of Innovations) under the Agreement (Agreement) No. 4174GS1 / 68618 dated July 23, 2021.

State of the art

Spinal muscular atrophy (SMA) is a severe autosomal recessive neurodegenerative disease characterized by progressive proximal muscle weakness and atrophy resulting from degeneration of alpha neurons in the anterior horns of the spinal cord. Despite the fact that the global estimated incidence of SMA does not exceed 1 case per 10,000 live births (Verhaart, I.E.C. Prevalence, incidence and carrier frequency of 5q linked spinal muscular atrophy a literature review / I.E.C. Verhaart, A. Robertson, I.J. Wilson , A. Aartsma-Rus, S. Cameron, C.C. Jones et al // Orphanet J Rare Dis. - 2017. - v. 12. - P. 15), this disease is the second most common autosomal recessive hereditary disease in the world and, moreover, is the most common monogenic disease leading to infant mortality. The frequency of occurrence of the mutant SMA allele varies from 1 in 38 to 1 in 72 cases, with a population average of 1 in 54 (Chen, T. New and Developing Therapies in Spinal Muscular Atrophy: From Genotype to Phenotype to Treatment and Where Do We Stand ? / T. Chen // Int J Mol Sci. - 2020. - v. 21. - P. 3297.).

The reason for the development of SMA in more than 95% of cases is a decrease in the level of expression of the protein of survival of motor neurons SMN (Survival of Motor Neuron), resulting from a homozygous deletion or conversion of the SMN1 gene. The genome contains an almost identical analogue of this gene - SMN2, however, it provides only 10-15% of functional protein synthesis (Blasco-Pérez, L. Beyond copy number: A new, rapid, and versatile method for sequencing the entire SMN2 gene in SMA patients / L. Blasco-Pérez, I. Paramonov, J. Leno, S. Bernal, L. Alias, P. Fuentes-Prior et al. // Hum Mutat. - 2021. - v. 42. - P. 787-795 .).

It is the SMN1 and SMN2 genes that are the main targets for the development of modern methods of therapy for spinal muscular atrophy. Currently, three drugs - Nusinersen (Spinraza; Biogen), Risdiplam (Eurysdy; Roche), and Onasemnogen abeparvovec (Zolgenema; Novartis) - are approved by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA). Nusinersen and Risdiplam affect the splicing of the mRNA of the CMN2 gene. The synonymous replacement of cytosine for thymine nucleotides in the SMN2 gene sequence forms a splicing enhancer and leads to the synthesis of a truncated protein variant - SMN-Δ7 (Schorling, D.C. Advances in Treatment of Spinal Muscular Atrophy - New Phenotypes, New Challenges, New Implications for Care / D.C. Schorling, A. Pechmann, J. Kirschner // J Neuromuscul Dis. - 2020. - v. 7. - P. 1-13.). By inhibiting splicing, these drugs can increase the level of functional SMN protein, but they have only a temporary effect and require annual administration (Blasco-Pérez, L. Beyond copy number: A new, rapid, and versatile method for sequencing the entire SMN2 gene in SMA patients / L. Blasco-Pérez, I. Paramonov, J. Leno, S. Bernal, L. Alias, P. Fuentes-Prior et al. // Hum Mutat. - 2021. - v. 42. - P. 787- 795.). The onasemnogen abeparvovec is a recombinant adeno-associated virus that carries a normal copy of the SMN1 cDNA under the control of a hybrid promoter consisting of a cytomegalovirus enhancer and an intron of the chicken β-actin gene. This drug provides restoration of SMN protein expression after a single injection, but it causes severe side effects in the form of liver failure (Chaytow, H. Spinal muscular atrophy: From approved therapies to future therapeutic targets for personalized medicine / H. Chaytow, K. M. E. Faller, Y. Huang, T. H. Gillingwater // Cell Rep Med. - 2021. - v. 2. P. 19.). Optimization of genetically engineered constructs containing adeno-associated virus genes and the SMN1 cDNA sequence by adding a neurospecific promoter of the SYN1 gene will reduce the hepatotoxicity of the drug, achieve remission in patients suffering from spinal muscular atrophy and avoid the development of severe side effects, especially in infants.

Disclosure of the essence of the invention

In the present invention, tissue-specific expression of the SMN1 gene is provided using the pAAVITR vector. The open reading frame of the SMN1 gene in the vector is located after the synapsin I SYN1 gene promoter. The synapsin I gene in the human body is associated with synaptic vesicles and is expressed only in neurons, which is provided by its neurospecific promoter. At the same time, the promoter of the synapsin I gene is one of the shortest among human gene promoters, which makes it convenient for use in genetic constructs. After the SMN1 gene, the genetic construct contains the polyadenylation signal of the bovine growth hormone bGH, which ensures polyadenylation of mRNA, which prolongs its lifetime by protecting the 3'-end from nucleases. All of these sequences are located between the inverted terminal repeats (ITR) of adeno-associated virus serotype 2. The presence of repeats ensures the replication and packaging of the functional part of the construct into capsid proteins and the assembly of virions carrying a therapeutic agent.

The presence of the β-lactamase (AmpR) gene in the vector makes it possible to produce it in Escherichia coli bacterial cells by selective selection using the antibiotic ampicillin. The expression of this gene occurs only in bacterial cells due to regulation by the promoter of the β-lactamase gene. Replication of the vector in prokaryotic cells is provided by the presence of the origin of replication ColEI.

The objective of the claimed invention is to create a gene therapy drug based on a recombinant adeno-associated virus serotype 2, carrying in its genome a normal open reading frame of the SMN1 gene under the control of the neurospecific promoter of the SYN1 synapsin gene as part of the pAAVTTR vector.

The problem is solved by the fact that a new genetic construct prSYN1_SMN1_AAVITR has been obtained, for which:

1. Using a polymerase chain reaction with overlapping primers, after reverse transcription, the synthesis of an open reading frame of a functional variant of the SMN1 gene with a size of 889 base pairs was performed, carrying at its ends restriction sites for restriction endonucleases HindIII and XbaI. The total RNA isolated from the IMR-32 human cell line, which is a mixture of two cell types: neuroblasts and fibroblasts, was used as a template. The sequencing of the synthesized open reading frame was verified by Sanger sequencing.

2. Synapsin gene promoter (SYN1) was synthesized using nested primer polymerase chain reaction under conditions of amplification of GC-rich templates using human cell line MRC-5, which are embryonic fibroblasts, as a genomic DNA template. A 448 bp product was obtained, carrying at its ends the recognition sites for restriction endonucleases KpnI and HindIII.

3. Assembly of the prSYN1_SMN1_AAVITR vector is characterized by the following stages:

a) Treatment of the plasmid obtained earlier on the basis of the construct pSpCas9(BB)-2A-GFP (РХ458) (Addgene #48138) with restriction endonucleases KpnI and XbaI, isolation of a fragment carrying the inverted terminal repeats of the adeno-associated virus, the bacterial origin ColE1 and the β-lactamase gene 3143 base pairs from agarose gel and its purification

b) Hydrolysis by restriction endonucleases HindIII and XbaI of the previously synthesized open reading frame of the SMN1 gene

c) Hydrolysis by restriction endonucleases KpnI and HindIII of the previously synthesized promoter of the SYN1 synapsin gene.

d) Ligation of all three obtained fragments with T-4 DNA ligase and subsequent cloning in bacterial cells of Escherichia coli strain NEB Stable.

e) Confirmation of the correct assembly of the prSYN1_SMN1_AAVITR vector using Sanger sequencing.

Technical result of the invention: a genetic construct carrying a functional variant of the SMN1 gene under the regulation of the neurospecific promoter of the SYN1 synapsin gene between the inverted terminal repeats of the adeno-associated virus serotype 2 was obtained, which ensures the production of recombinant adeno-associated viruses for delivery of the functional part of the vector to cells in vivo.

The invention is illustrated by the figure and the list of sequences used.

Brief description of the drawings

The figure shows a diagram of the prSYN1_SMN1_AAVITR vector. Between the inverted terminal repeats of the ITR adeno-associated virus, there is a functional open reading frame of the SMN1 gene under the control of the neurospecific promoter of the SYN1 synapsin gene.

Implementation of the invention

The method is carried out as follows:

Obtaining genetic constructs. cDNA of the SMN1 gene (SEQ ID NO: 1) was obtained based on total mRNA obtained from total RNA of IMR-32 cells isolated by phenol-chloroform extraction. Reverse transcription was performed using M-MuLV-RH reverse transcriptase (Biolabmix) and three primer variants SMN1_1-R (SEQ ID NO: 3), SMN1_2-R (SEQ ID NO: 4), SMN1_3-R (SEQ ID NO: 5 ). Amplification of the SMN7 coding sequence was performed using a nested PCR system and primers SMN1_Kozak_KpnI-F (SEQ ID NO: 6), SMN1_dell-F (SEQ ID NO: 7), SMN1_dell-R (SEQ ID NO: 8), SMN1_del2-F (SEQ ID NO: 9), SMN1_del2-R (SEQ ID NO: 10), SMN1_XbaI-R (SEQ ID NO: 11) with DNA polymerase Q5 (NEB). The synthesized fragment was cut with restriction endonucleases HindIII and XbaI (SybEnzyme).

The synthesis of the Syn1 promoter (SEQ ID NO: 2) was carried out on the basis of the DNA material isolated from the MRC-5 cell culture using phenol-chloroform extraction and alcohol precipitation. In the synthesis, primers Syn1_KpnI-F (SEQ ID NO: 12) and Syn1_HindIII-R (SEQ ID NO: 13) and DNA polymerase Q5 (NEB) were used with the addition of dimethyl sulfoxide. The synthesized fragment was cut with restriction endonucleases HindIII and KpnI (SybEnzyme).

The pAAVITR vector, previously derived from the construct pSpCas9(BB)-2A-GFP (РХ458) (Addgene #48138), was digested with restriction endonucleases XbaI and KpnI (SybEnzyme) and purified by agarose gel extraction using the QIAquick Gel Extraction Kit (Qiagen)

Ligation of the three resulting fragments was performed using T4 phage DNA ligase (Thermo Fisher Scientific). The required number of fragments for the ligation reaction was calculated using the NEBioCalculator (NEB) online calculator. For the successful completion of the reaction, the ratio of copies of the DNA inserts relative to the copies of the vector DNA was 7 to 1, while 100 ng of the fragment was taken for the reaction. The ligation mixture was transformed into competent NEB Stable (NEB) cells, colonies were screened, grown in overnight culture, plasmid DNA was isolated from them and checked using restrictive analysis. One sample was sequenced and checked if the sequence fully matched the predicted (SEQ ID NO: 1 and SEQ ID NO: 2).

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Sequence listing

<110> TRANSGEN LIMITED LIABILITY COMPANY (OBSCHESTVO S

OGRANICHENNOI OTVETSTVENNOSTYU TRANSGEN)

<120> Genetic construct adapted to deliver the SMN1 gene

human with adeno-associated virus serotype 2 for

ensuring neurospecific expression

<160> 13

<210> 1

<211> 889

<212> SMN1 DNA open reading frame sequence

<213> H. sapiens

<400> 1 atg gcg atg agc agc ggc ggc agt ggt ggc ggc gtc ccg gag cag

gag gat tcc gtg ctg 60

ttc cgg cgc ggc aca ggc cag agc gat gat tct gac att tgg gat gat aca

gca ctg ata 120

aaa gca tat gat aaa gct gtg gct tca ttt aag cat gct cta aag aat ggt

gac att tgt 180

gaa act tcg ggt aaa cca aaa acc aca cct aaa aga aaa cct gct aag aag

aat aaa agc 240

caa aag aag aat act gca gct tcc tta caa cag tgg aaa gtt ggg gac aaa

tgt tct gcc 300

att tgg tca gaa gac ggt tgc att tac cca gct acc att gct tca att gat

ttt aag aga 360

gaa acc tgt gtt gtg gtt tac act gga tat gga aat aga gag gag caa aat

ctg tcc gat 420

cta ctt tcc cca atc tgt gaa gta gct aat aat ata gaa caa aat gct caa

gag aat gaa 480

aat gaa agc caa gtt tca aca gat gaa agt gag aac tcc agg tct cct gga

aat aaa tca 540

gat aac atc aag ccc aaa tct gct cca tgg aac tct ttt ctc cct cca cca

ccc ccc atg 600

cca ggg cca aga ctg gga cca gga aag cca ggt cta aaa ttc aat ggc cca

cca ccg cca 660

ccg cca cca cca cca ccc cac tta cta tca tgc tgg ctg cct cca ttt cct

tct gga cca 720

cca ata att ccc cca cca cct ccc ata tgt cca gat tct ctt gat gat gct

gat gct ttg 780

gga agt atg tta att tca tgg tac atg agt ggc tat cat act ggc tat tat

atg ggt ttc 840

aga caa aat caa aaa gaa gga agg tgc tca cat tcc tta aat taa

<210> 2

<211> 448

<212> DNA SYN1 promoter sequence

<213> H. sapiens

<400> 1 agt gca agt ggg ttt tag gac cag gat gag gcg ggg tgg ggg tgc

cta cct gac gac cga 60

ccc cga ccc act gga caa gca ccc aac ccc cat tcc cca aat tgc gca tcc

cct atc aga 120

gag ggg gag ggg aaa cag gat gcg gcg agg cgc gtg cgc act gcc agc ttc

agc acc gcg 180

gac agt gcc ttc gcc ccc gcc tgg cgg cgc gcg cca ccg ccg cct cag cac

tga agg cgc 240

gct gac gtc act cgc cgg tcc ccc gca aac tcc cct tcc cgg cca cct tgg

tcg cgt ccg 300

cgc cgc cgc cgg ccc agc cgg acc gca cca cgc gag gcg cga gat ag g ggg

gca cgg gcg 360

cga cca tct gcg ctg cgg cgc cgg cga ctc agc gct gcc tca gtc tgc ggt

ggg cag cgg 420

agg agt cgt gtc gtg cct gag agc gca g

<210> 3

<211> 20

<212> DNA

<213> artificial sequence

<220> reverse primer

<223> SMN1_1-R

<400> 1 gcc aca tac gcc tca cat ac

<210> 4

<211> 25

<212> DNA

<213> artificial sequence

<220> reverse primer

<223> SMN1_2-R

<400> 1 gat tct gta aca ttt tgc cac ata c

<210> 5

<211> 21

<212> DNA

<213> artificial sequence

<220> reverse primer

<223> SMN1_3-R

<400> 1 aca atg aac agc cat gtc cac

<210> 6

<211> 35

<212> DNA

<213> artificial sequence

<220> direct primer

<223> SMN1_Kozak_KpnI-F

<400> 1 taa gca ggt acc ttt gct atg gcg atg agc agc gg

<210> 7

<211> 22

<212> DNA

<213> artificial sequence

<220> direct primer

<223> SMN1_del1-F

<400> 1 gtc taa aat tca atg gcc cac c

<210> 8

<211> 22

<212> DNA

<213> artificial sequence

<220> reverse primer

<223> SMN1_del1-R

<400> 1 ggt ggg cca ttg aat ttt aga c

<210> 9

<211> 26

<212> DNA

<213> artificial sequence

<220> direct primer

<223> SMN1_del2-F

<400> 1 cta tta tat ggg ttt cag aca aaa tc

<210> 10

<211> 27

<212> DNA

<213> artificial sequence

<220> reverse primer

<223> SMN1_del2-R

<400> 1 gat ttt gtc tga aac cca tat aat agc

<210> 11

<211> 30

<212> DNA

<213> artificial sequence

<220> reverse primer

<223> SMN1_XbaI-R

<400> 1 taa gca tct aga gtg ctg ctc tat gcc agc

<210> 12

<211> 33

<212> DNA

<213> artificial sequence

<220> direct primer

<223> Syn1_KpnI-F

<400> 1 taa gca ggt acc agt gca agt ggg ttt tag gac

<210> 13

<211> 29

<212> DNA

<213> artificial sequence

<220> reverse primer

<223> Syn1_HindIII-R

<400> 1 taa gca aag ctt ctg cgc tct cag gca cg

<---

Claims (8)

Recombinant plasmid prSYN1_SMN1_AAVITR designed to package the sequence of the functional open reading frame of the SMN1 gene under the control of the synapsin I SYN1 gene promoter into virions of the recombinant adeno-associated virus serotype 2 to ensure the expression of the SMN1 gene in human neuronal cells, which has a size of 4516 base pairs, contains the target gene, which is an open reading frame of the SMN1 gene, having the nucleotide sequence of SEQ ID NO: 1, 885 base pairs long, under the control of the SYN1 promoter, having the nucleotide sequence of SEQ ID NO: 2, 448 base pairs long, and consists of the following elements: the β-lactamase gene, having coordinates from 1298 to 2158 bp, under the control of a promoter with coordinates from 1193 to 1297 bp to ensure the selective selection of Escherichia coli bacteria using the antibiotic ampicillin; replication origin - ori, having coordinates from 3605 to 4489 base pairs, to ensure vector replication in Escherichia coli bacterial cells; inverted terminal repeats of adeno-associated virus serotype 2 with coordinates from 2998 to 3138 base pairs and from 241 to 381 base pairs, providing packaging of the target SMN1 gene under the control of the SYN1 promoter into adeno-associated virus virions; And polyadenylation signal bGH poly(A) signal, having coordinates from 25 to 232 bp, providing mRNA polyadenylation.