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WO2024038287A1 - Thérapie génique pour le traitement d'une déficience en argininosuccinate lyase - Google Patents

Thérapie génique pour le traitement d'une déficience en argininosuccinate lyase Download PDF

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Publication number
WO2024038287A1
WO2024038287A1 PCT/GB2023/052174 GB2023052174W WO2024038287A1 WO 2024038287 A1 WO2024038287 A1 WO 2024038287A1 GB 2023052174 W GB2023052174 W GB 2023052174W WO 2024038287 A1 WO2024038287 A1 WO 2024038287A1
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Prior art keywords
sequence
vector
asl
composition
derivative
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Inventor
Julien BARUTEAU
Paul GISSEN
John Counsell
Loukia TOURAMANIDOU
Dany PEROCHEAU
Simon Waddington
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UCL Business Ltd
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UCL Business Ltd
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Priority claimed from GBGB2212092.7A external-priority patent/GB202212092D0/en
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Priority to EP23761975.4A priority Critical patent/EP4573193A1/fr
Publication of WO2024038287A1 publication Critical patent/WO2024038287A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/00041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/00043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y403/00—Carbon-nitrogen lyases (4.3)
    • C12Y403/02—Amidine-lyases (4.3.2)
    • C12Y403/02001—Argininosuccinate lyase (4.3.2.1)

Definitions

  • the present invention relates to a gene therapy transgene cassette for the treatment of Argininosuccinate Lyase Deficiency (ASLD).
  • ASLD Argininosuccinate Lyase Deficiency
  • the gene therapy preferably utilises a lentiviral gene therapy transgene cassette which is an integrating vector which can be used to successfully correct ASLD in neonates, children and teenagers.
  • urea cycle is an essential liver pathway enabling the removal of neurotoxic ammonia, produced by catabolism of amino acids.
  • Argininosuccinate Lyase Deficiency is the second most common urea cycle disorder (UCD), with prevalence of 1/70,000 to 1/100,000 live births (1). This prevalence is much higher in the Middle East (e.g. Saudi Arabia) where the prevalence can be as high as 1/15,000. This is a much rarer condition in Asia (1/500,000 to 1/1 ,000,000). Based on these data, it is expected that 6-7 newborns per year in the UK will have ASLD and 40 per year in Europe and a similar number in the USA. In Saudia Arabia, it is expected that 35 new born children every year will have ASLD.
  • Patients with ASLD may present with hyperammonaemia neonatally (early-onset), or later in life (late-onset), and require intensive care to restore life-compatible ammonia levels. Delay in appropriate management increases the risk of neurologic sequelae and death. Patients suffer recurrent hyperammonaemic crisis causing high rates of mortality and neurodisability, with learning difficulties, behavioural problems and epilepsy. The standard of care relies on ammonia scavengers, protein-restricted diet and arginine supplementation but this does not prevent recurrent hyperammonaemic decompensations (2). Severe patients undergo liver transplantation, a curative procedure balanced by lifelong immunosuppression and procedure-related complications. The UCD lifetime cost is now estimated over £ million (3).
  • the only approved therapies are pharmaceutically active ammonia scavengers approved for hyperammonaemia in urea cycle defects i.e. sodium benzoate, sodium phenylbutyrate, glycerol phenylbutyrate. Patients usually receive L-arginine supplementation and follow a protein restricted diet.
  • therapies only seek to manage ASLD rather than being corrective.
  • Gene therapy has previously been proposed for the treatment of ASLD and AAV gene therapy for ASLD has shown a successful correction of the disease in adult treated ASLD mice but only a mild correction in neonatally treated mice (4). This is due to the rapid liver growth after neonatal injection.
  • the transgene cassette is lost during the first 4 weeks of life when the liver doubles its size 5 times (5) and therefore a suitable therapy is still needed, not least to be able to treat neonatal patients where a corrective therapy would have the greatest effect.
  • liver transplantation which requires lifelong immunosuppression, has procedure-related morbidity and mortality and shortage of donors. This procedure is rarely done in neonates, and is usually performed for older children or teenagers when they develop a chronic liver disease like liver fibrosis or have repeated hyperammonaemia due to metabolic instability.
  • composition comprising an optimised human Argininosuccinate Lyase (ASL) nucleic acid sequence of SEQ ID No. 9 or a derivative sequence having at least about 94% sequence identity thereof.
  • ASL optimised human Argininosuccinate Lyase
  • the derivative sequence may have at least about 95% sequence identity thereof, at least about 96% sequence identity thereof, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity thereof to SEQ ID No. 9.
  • the derivative sequence may have at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereof to SEQ ID No. 9.
  • sequence identity is determined by comparing two aligned substantially complementary sequences over their length and overall identity is expressed as a percentage.
  • the measurement of nucleotide sequence identity is well known in the art, using specialist computer programs such as “BLAST”.
  • the nucleic acid sequence may be a DNA, RNA, mRNA, cDNA, genomic DNA or PNA and may be recombinant or synthetic which differs from the nucleic acid sequence found in nature. It may be single stranded or double stranded.
  • the nucleic acid sequence will encode the optimised ASL nucleic acid sequence of SEQ ID No. 9, or derivative sequence thereof having at least about 94% sequence identity.
  • the nucleic acid sequence may be derived by cloning, for example using standard molecular cloning techniques including restriction digestion, ligation, gel electrophoresis (for example as described in Sambrook et al; Molecular Cloning: A laboratory manual, Cold Spring Harbour laboratory Press).
  • the nucleic acid sequence may be isolated or amplified using PCR technology. Such technology may employ primers based upon the sequence of the nucleic acid sequence to be amplified. With the sequence information provided, the skilled person can use available cloning techniques to produce a nucleic acid sequence or vector suitable for transduction into a cell.
  • the nucleic acid sequence may alternatively have been generated de novo by DNA synthesis, which can be performed using routine procedures in the field of DNA synthesis.
  • the optimised ASL nucleic acid sequence may be optimised in a number of ways so as to enable enhanced expression or activity.
  • the sequence may have been codon optimised by selecting codons most common in human cells and/or reducing one or more secondary structures and hairpins which may arise in subsequently formed mRNA.
  • the optimised ASL nucleic acid sequence may be under the control of a suitable promoter. It is preferred that the optimised ASL nucleic acid sequence is under the control of a Liver-specific promoter 1 (LP1) promoter. In some embodiments, it is preferred that the LP1 promoter has the sequence of SEQ ID No. 8 or a derivative sequence having at least about 90% sequence identity thereof. If a derivative sequence of the SEQ ID No. 8 is employed, the derivative sequence may have at least about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereof to SEQ ID No. 8.
  • LP1 promoter has the sequence of SEQ ID No. 8 or a derivative sequence having at least about 90% sequence identity thereof. If a derivative sequence of the SEQ ID No. 8 is employed, the derivative sequence may have at least about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,
  • the WPRE element may have the sequence of SEQ ID No. 10 or a derivative sequence thereof having at least about 90% sequence identity. If a derivative sequence of the SEQ ID No. 10 is employed in the vector, the derivative sequence may have at least about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereof to SEQ ID No. 10.
  • the LP1 promoter and WPRE element assist with mRNA stability and neither have been previously utilised in a lentiviral vector.
  • composition of the present invention is preferably suitable for use in vivo or in vitro, and is preferably suitable for use in a human.
  • optimised human ASL nucleic acid sequence was shown by the present inventors to advantageously increase expression of ASL by 2-fold. This is particularly important for vectors such as lentiviral vectors, where lower titres of these vectors are employed due to lower levels of vectors produced during manufacturing.
  • the composition may be formulated into a therapy for targeting neonatal infants, young children and teenagers affected by ASLD and will act like a liver replacement strategy.
  • patients will not require any further ammonia scavengers or be on a protein restricted diet and they will not be at risk of hyperammonaemic decompensation anymore for decades, with no need for re injection or immunosuppression.
  • the optimised ASL sequence is incorporated into an integrating and/or lentiviral vector and/or a gamma retroviral vector.
  • the optimised ASL sequence is incorporated into a lentiviral (LV) vector.
  • the vector comprises a pCCL backbone.
  • the vector will preferably comprise one or more regulatory sequences to direct expression of the optimised ASL nucleic acid sequence, or derivative sequence thereof.
  • a regulatory sequence may include a promoter operably linked to the nucleic acid sequence, an enhancer, a transcription termination signal, a polyadenylation sequence, an origin of replication, a nucleic acid restriction site, and a homologous recombination site.
  • a vector may also include a selectable marker, for example to determine expression of the vector in a growth system (for example a bacterial cell) or in a target neural cell.
  • operably linked means that the nucleic acid sequence is functionally associated with the sequence to which it is operably linked, such that they are linked in a manner such that they affect the expression or function of one another.
  • a nucleic acid sequence operably linked to a promoter will have an expression pattern influenced by the promoter.
  • the composition may be for use in the treatment of a disease or condition attributable to Argininosuccinate Lyase Deficiency (ASLD).
  • ASLD Argininosuccinate Lyase Deficiency
  • composition may be for use in the preparation of a medicament for the treatment of a disease or condition attributable to ASLD.
  • a disease or condition attributable to ASLD may selected from one or more of the following commonly associated diseases or conditions: hyperammonaemia, arterial hypertension, developmental delay and chronic liver disease.
  • Neonatal treatment may be defined as the administration of the composition of the invention within 8 hours, the first 12 hours, the first 24 hours, or the first 48 hours of delivery. Neonatal delivery may be within the period of about 12 hours to about 1 week, 2 weeks, 3 weeks, or about 1 month, or after about 24 hours to about 48 hours. Due to rapid turnover of liver cells, neonatal therapy is desirably followed by readministration at about 3 months of age, about 6 months, about 9 months, or about 12 months. More than one re-administration may be desirable.
  • composition is formulated for intravenous infusion and/or intra-arterial delivery.
  • the composition may be a liquid or a solid, for example a powder, gel, or paste.
  • a composition is a liquid, preferably an injectable liquid.
  • Such an injectable liquid will preferably be suitable for hepatic artery infusion administration.
  • the composition may also comprise one or more excipients and such excipients will be known to persons skilled in the art.
  • composition may incorporate or be administered in conjunction (either sequentially or simultaneously) with (or co-administered with) an immunosuppressant.
  • immunosuppressants may be selected from one or more of the following: tacrolimus, mycofenolate mofetil and prednisolone. The skilled addressee will understand that other immunosuppressants may also be employed.
  • an integrating and/or lentiviral and/or gamma retroviral vector comprising an expression cassette which comprises a Argininosuccinate Lyase (ASL) nucleic acid sequence or a derivative sequence encoding a functional human argininosuccinate lyase.
  • ASL Argininosuccinate Lyase
  • An "expression cassette” refers to a nucleic acid molecule which comprises the ASL nucleic acid sequence, promoter, and may include other regulatory elements.
  • the expression cassette may be packaged into the capsid of a viral vector.
  • Such an expression cassette for generating a viral vector may contain the ASL nucleic acid sequence flanked by packaging signals of the viral genome and other expression control sequences which are known in the art.
  • the integrating and/or lentiviral vector may comprise an optimised human Argininosuccinate Lyase (ASL) nucleic acid sequence of SEQ ID No. 9 or a derivative sequence having at least about 94% homology thereof.
  • ASL Argininosuccinate Lyase
  • the vector is a lentiviral vector. In another embodiment, the vector is a gamma retroviral vector.
  • the derivative sequence may have at least about 95% sequence identity thereof, at least about 96% sequence identity thereof, at least about 97% sequence identity thereof, at least about 98% sequence identity thereof or at least about 99% sequence identity thereof to SEQ ID No. 9.
  • the derivative sequence may have at least about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereof to SEQ ID No. 9.
  • the optimised ASL nucleic acid sequence in the vector is under the control of a Liver-specific promoter 1 (LP1) promoter.
  • the LP1 promoter may have the sequence of SED ID No. 8 or a derivative sequence thereof having at least about 90% sequence identity. If a derivative sequence of the SEQ ID No. 8 is employed in the vector, the derivative sequence may have at least about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereof to SEQ ID No. 8.
  • the WPRE element may have the sequence of SEQ ID No. 10 or a derivative sequence thereof having at least about 90% sequence identity. If a derivative sequence of the SEQ ID No. 10 is employed in the vector, the derivative sequence may have at least about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereof to SEQ ID No. 10.
  • the LP1 promoter and WPRE element assist with mRNA stability and neither have been previously utilised in a lentiviral vector.
  • the vector may further comprise one or more of the following elements: a cytomegalovirus (CMV) enhancer, a cytomegalovirus (CMV) promoter, a 5’ long terminal repeats (LTR), a human immunodeficiency virus type 1 packaging signal (HIV-1 ip), a Rev/Rev-responsive element (RRE), a central polypurine tract/central termination sequence (cPPT/CTS), a WPRE sequence, 3’ long terminal repeats (LTR), a Simian virus 40 PolyA (SV40 polyA) signal, Simian virus 40 (SV40) ori, a F1 origin of replication (ori), NeoR/KanR, and a Origin of replication Ori.
  • CMV cytomegalovirus
  • CMV cytomegalovirus
  • CMV cytomegalovirus
  • LTR long terminal repeats
  • HSV-1 ip human immunodeficiency virus type 1 packaging signal
  • RRE Rev/Rev-responsive element
  • the vector may comprise one or more of the following elements upstream of the liver specific promoter LP1 : a cytomegalovirus (CMV) enhancer; a cytomegalovirus (CMV) promoter; a 5’ long terminal repeats (LTR); a human immunodeficiency virus type 1 packaging signal (HIV-1 ip); a Rev/Rev-responsive element (RRE); and a central polypurine tract/central termination sequence (cPPT/CTS).
  • CMV cytomegalovirus
  • CMV cytomegalovirus
  • CMV cytomegalovirus
  • LTR long terminal repeats
  • HSV-1 ip human immunodeficiency virus type 1 packaging signal
  • RRE Rev/Rev-responsive element
  • CPS central polypurine tract/central termination sequence
  • the vector may comprise one or more of the following elements downstream of optimised ASL gene: 3’ long terminal repeats (LTR); a Simian virus 40 PolyA (SV40 polyA) signal; Simian virus 40 (SV40) ori; a F1 origin of replication (ori); NeoR/KanR; and Origin of replication Ori.
  • LTR long terminal repeats
  • SV40 polyA Simian virus 40 PolyA
  • SV40 Simian virus 40
  • ori SV40
  • ori origin of replication
  • NeoR/KanR NeoR/KanR
  • Origin of replication Ori Origin of replication Ori.
  • the elements will preferably comprise:
  • One or more derivative sequence of any of SEQ ID Nos 2 - 7 and/or SEQ ID Nos 11 - 16 may be employed with sequence identity in the range of about 90 - 99%. Additionally, the vector may or may not have coding or non-coding intervening sequences between each and every element.
  • the vector may be for use in the treatment of a disease or condition attributable to Argininosuccinate Lyase Deficiency (ASLD).
  • ASLD Argininosuccinate Lyase Deficiency
  • the vector may be for use in the preparation of a medicament for the treatment of a disease or condition attributable to ASLD.
  • the disease or condition attributable to ASLD may selected from one or more of the following commonly associated diseases or conditions: hyperammonaemia, arterial hypertension, developmental delay and chronic liver disease.
  • the vector is formulated for intravenous infusion and/or intra-arterial delivery.
  • a vector is formulated in the form of a liquid, preferably an injectable liquid.
  • a liquid preferably an injectable liquid.
  • an injectable liquid will preferably be suitable for intravenous infusion administration.
  • the vector may incorporate or be administered in conjunction (either sequentially or simultaneously) with (or co-administered with) an immunosuppressant.
  • immunosuppressants may be selected from one or more of the following: tacrolimus, mycofenolate mofetil and prednisolone. The skilled addressee will understand that other immunosuppressants may also be employed.
  • kits of parts for use in the treatment of an individual suffering from a disease or condition attributable to Argininosuccinate Lyase Deficiency (ASLD), the kit comprising: a) a composition or a vector as herein above described; and b) one or more catheters or syringes for intravenous infusion of the said composition or vector.
  • ASLD Argininosuccinate Lyase Deficiency
  • the composition or vector is in a buffer solution.
  • the kit may further comprise an immunosuppressant.
  • a viral or not viral vector is used to deliver the components of the genome editing system.
  • the vector is capable of delivering one or more components (e.g. , the guide RNA, donor template, and endonuclease) of the genome editing system, such as CRISPR-Cas9.
  • a combination or dual vector system is provided to deliver one or more components of the CRISPR system when being co-administered to an individual.
  • the vectors delivering donor template which are gene fragments may be configured so that the donor template is inserted upstream of the ASL gene mutation or phenotype to be corrected.
  • a vector may include a full-length sequence that can replace the defective ASL with an optimised ASL nucleic acid sequence of SEQ ID No. 9 or derivative sequence having at least about 94% sequence identity thereof.
  • a dual vector system may be provided which comprises (a) a gene editing vector which comprises an optimised ASL nucleic acid sequence of SEQ ID No. 9 or derivative sequence having at least about 94% sequence identity thereof under control of regulatory sequences which direct its expression in a target cell (e.g., a hepatocyte) comprising a targeted defective ASL gene which has one or more mutations and (b) a targeting vector comprising a sequence specifically recognized by the editing enzyme and donor template, wherein the donor template comprises nucleic acid sequences which replaces at least one of the mutations in the targeted defective ASL gene.
  • a gene editing vector which comprises an optimised ASL nucleic acid sequence of SEQ ID No. 9 or derivative sequence having at least about 94% sequence identity thereof under control of regulatory sequences which direct its expression in a target cell (e.g., a hepatocyte) comprising a targeted defective ASL gene which has one or more mutations
  • a targeting vector comprising a sequence specifically recognized by the editing enzyme and donor template, wherein the donor template comprises
  • Figure 1 is a plasmid map of the CCL-LP1-co.hASL plasmid (8419bp) which was used in the experiments described in the examples.
  • Figure 2 are graphs and images showing that the systemic injection of LV.cohASL sustainably improves the macroscopic phenotype of Asl Neo/Neo mice after neonatal injection.
  • C Liver/body weight ratio.
  • Figure 3 are graphs showing the correction of the urea cycle after gene therapy.
  • A Plasma ammonia,
  • B argininosuccinic acid,
  • C L-citrulline and
  • D L-arginine from dried blood spots in 3 month-old mice.
  • E Urine orotate
  • F Liver ASL activity.
  • Figure 4 are images and graphs showing the correction of the urea cycle after gene therapy.
  • A representative images of hASL immunostaining in liver
  • B computational calculation of hASL stained area in liver from 10 representative images for each animal
  • C liver hASL western blot
  • D western blot analysis in WT, LV.GFP and LV.cohASL treated AslNeo/Neo mice at harvest (statistical analysis of log-transformed data).
  • Figure 5 are graphs showing the in vitro enhanced efficacy of codon-optimised hASL transgene versus WT hASL
  • A Overexpression of cohASL and hASL plasmids compared to endogenous expression in Huh7 cells
  • B Overexpression of coASL and ASL lentiviral vectors compared to endogenous expression in Huh7 cells at different MOI at 24 hours post-transfection
  • C Overexpression of coASL and ASL lentiviral vectors compared to endogenous expression in Huh7 cells over time at MOI 20 and 70.
  • FIG. 6 shows photographs of mice who have received (A) Neonatal lentiviral gene therapy after 3 months of age (two mice are shown: Wild Type (WT) and Lentiviral Gene Therapy (GT)); and (B) Neonatal AAV gene therapy after 6 months of age (three mice are shown: AAV WT, WT and untreated).
  • A Neonatal lentiviral gene therapy after 3 months of age
  • two mice are shown: Wild Type (WT) and Lentiviral Gene Therapy (GT)
  • B Neonatal AAV gene therapy after 6 months of age
  • Figure 7 are graphs showing the (A) survival and (B) growth of the following mice: WT, Untreated Asl Neo/Neo ; neonatal LV Asl Neo/Neo ; and neonatal AAV Asl Neo/Neo .
  • Figure 8 are graphs showing the levels of (A) orotic acid (orotate), (B) arginine (citrulline) and (C) argininosuccinic acid at 3 months in the following mice: WT, Untreated Asl Neo/Neo and neonatal LV Asl Neo/Neo and neonatal AAV Asl Neo/Neo (** p ⁇ 0.01 , *** p ⁇ 0.001 , **** p ⁇ 0.0001 , ns not significant).
  • Figure 9 graphs showing the level of arginine at 3 months in the following mice: (A) WT, Untreated Asl Neo/Neo , neonatal LV Asl Neo/Neo ; and (B) WT, Untreated Asl Neo/Neo , neonatal AAV not significant).
  • Figure 10 graphs showing the level of arginine as an indicator of liver ASL activity (A) at 3 months in the following mice: WT, Untreated Asl Neo/Neo , LV Asl Neo/Neo and (B) at 9 months in the following mice: WT, Untreated Asl Neo/Neo , AAV Asl Neo/Neo (* p ⁇ 0.05, *** p ⁇ 0.001 , **** p ⁇ 0.0001 , ns not significant).
  • Figure 11 is a schematic diagram showing the protocol to assess the safety profile of the LV.cohASL in a murine mouse model and human hepatocytes.
  • Figure 12 are graphs showing the safety of the LV.cohASL in vivo.
  • B Vector genome copy number in liver samples from LV.cohASL- and PBS-treated CD1 mice (unpaired two-tailed Student’s t test; * p ⁇ 0.05, ** p ⁇ 0.01 , *** p ⁇ 0.001 , ns not significant). Graphs show means ⁇ SD.
  • C Biodistribution of vector copies per cell in five randomly selected LV.cohASL- treated CD1 mice.
  • Figure 13 are graphs showing (A) the number of integrations, and (B) the Shannon Diversity Index per sample. (C) The ratio of frequency of identification for each ISA (%) out of total ISA events.
  • Vector copy number was analysed on every sample via droplet digital PCR (BioRad QX200 system).
  • the LV primers were designed over the Psi region while reference control assays were designed over the Titin gene for the murine tissues and over the SPIDR gene for the human samples.
  • the psi copies were divided by the titin copies and divided by two to infer the average VCN/diploid cell.
  • LM-PCR linker mediated PCR
  • gDNA was harvested from the murine samples 9 months after IV vector transduction.
  • gDNA from human primary hepatocytes was extracted one week after transduction. Briefly, ca. 250 ng of gDNA were fragmented and a double-stranded linker DNA ligated using the NEB Next Ultra II FS DNA Library Prep Kit and NEB Next Ultra II Ligation Master Mix and Ligation Enhancer (New England Biolabs). Specific primers were utilized to perform the LM-PCR from the viral LTR.
  • PCR reactions were purified and barcoded with NEB Next Multiplex Oligos for Illumina (New England Biolabs).
  • AMPpure XP beads purification (Beckman Coulter) was set at O.7X volume to select fragments >180bp.
  • the libraries were finally sequenced through the Illumina NovaSeq platform and analysed with the bioinformatic pipeline (htps://github.com/AG-Boerries/CAST-Seq) calling as true IS events reporting more than 3 reads then deduplicated and quantified based on the unique molecular signature derived by the sonic abundance method.
  • An in silico random IS library of 10,000 events was generated to perform a statistical comparison over the genetic features distribution and to control the clustering thresholds.
  • Lentiviral integrations can label every single cell in a unique manner as its landing site is semi-random with a slight preference for actively expressed genes (6).
  • a clonal expansion burst will result in an unusual quantification of one specific integration site while an even cell duplication across the whole bulk population will return an even quantification across all the mapped integrations.
  • the Shannon Diversity Index (EH) helps to objectively measure the diversity of species in a population.
  • Murine liver samples were collected at 1 and 9 months after treatment to potentially address any potential genotoxic outcome.
  • Example 1 Lentiviral Gene Therapy Vector (LV.cohASL)
  • a lentiviral gene therapy vector (LV.cohASL) was produced and assessed in a ASLD mouse model.
  • the vector comprises a pCCL backbone, a liver-specific promoter LP1 (ApoE enhancer, human a1 antitrypsin promoter) (SEQ ID No. 8), a codon-optimised version of the human ASL gene (SEQ ID No. 9).
  • the sequence of the vector incorporating the codon optimised human ASL gene is provided in SEQ ID No. 1.
  • This vector was produced by triple transfection in HEK293T cells and titrated by qPCR targeting WPRE.
  • a plasmid map of the vector is shown in Figure 1.
  • the vector Upstream of the liver specific promoter LP1 , the vector also comprised a cytomegalovirus (CMV) enhancer (SEQ ID No. 2), a cytomegalovirus (CMV) promoter (SEQ ID No. 3), a 5’ long terminal repeats (LTR) (SEQ ID No. 4), a human immunodeficiency virus type 1 packaging signal (HIV-1 i ) (SEQ ID No. 5), a Rev/Rev-responsive element (RRE) (SEQ ID No. 6), a central polypurine tract/central termination sequence (cPPT/CTS) (SEQ ID No. 7).
  • a cytomegalovirus (CMV) enhancer SEQ ID No. 2
  • CMV cytomegalovirus
  • CMV cytomegalovirus
  • LTR long terminal repeats
  • HSV-1 i human immunodeficiency virus type 1 packaging signal
  • RRE Rev/Rev-responsive element
  • cPPT/CTS central polypurine tract
  • LTR long terminal repeats
  • SV40 polyA Simian virus 40 PolyA signal
  • SV40 Simian virus 40
  • ori SV40 ori
  • F1 origin of replication ori
  • NeoR/KanR SEQ ID No. 15
  • Origin of replication Ori SEQ ID No. 16
  • LV.cohASL Lentiviral vector
  • GFP green fluorescent protein
  • mice injected with LV.cohASL showed rescue of survival sustainably improves the macroscopic phenotype of Asl Neo/Neo mice after neonatal injection and had a 100 % survival rate over 80 days, which compared to a greatly reduced (less than 15%) survival rate for those mice having being injected with the LV.GFP vector.
  • Figure D are images of mice at week 4 which show that those mice injected with LV.GFP were much smaller than WT, whereas mice injected with LV.cohASL were comparable in size to WT and were also comparable in size to WT at week 8 and had similar fur patterns.
  • Figure 2B shows that mice injected with LV.cohASL had achieved normalisation of growth which was similar to that of WT, whereas mice included with LV.GFP weighed substantially less.
  • Figure 2C shows that the liver/body weight ratio was more similar in the WT and LV.cohASL mice when compared to the mice injected with LV.GFP.
  • FIG. 3A the plasma ammonia levels were shown to be normalised to WT in those mice injected with LV.cohASL, whereas those mice injected with LV.GFP had elevated ammonia levels.
  • Figure 3B the level of argininosuccinic acid in dried blood spots was shown to be normalised to WT in those mice injected with LV.cohASL, whereas those mice injected with LV.GFP had elevated argininosuccinic acid levels.
  • Figure 4A shows images of hASL immunostaining in liver samples and show that comparable ASL activity was found in the liver of WT mice and mice injected with LV.cohASL. In comparison, no ASL activity was found in those mice injected with LV.GFP.
  • Figure 4B shows the computational calculation of hASL stained area in liver from 10 representative images for each animal and shows that whilst ASL activity was found in mice injected with LV.cohASL, no such activity was found in those mice injected with LV.GFP.
  • Figure 4C shows liver hASL western blot
  • Figure 4D shows western blot analysis in WT, LV.GFP and LV.cohASL treated Asl Neo/Neo mice at harvest. Again, whilst comparative ASL activity to WT was found in mice injected with LV.cohASL, no such activity was found in those mice injected with LV.GFP.
  • Figure 5 shows graphs illustrating the in vitro enhanced efficacy of codon-optimised hASL transgene versus WT hASL.
  • the codon-optimised hASL has 81 .4% sequence identity with the WT hASL.
  • Figure 5A shows that there is an overexpression of cohASL and hASL plasmids compared to endogenous expression hASL in Huh7 cells.
  • Figure 5B also shows the overexpression of coASL and ASL lentiviral vectors compared to endogenous expression in Huh7 cells at different MOI at 24 hours post-transfection
  • Figure 5C also shows the overexpression of coASL and ASL lentiviral vectors compared to endogenous expression in Huh7 cells over time at MOI 20 and 70 and clearly shows that the codon optimised hASL shows a 2-fold increase in expression levels when compared to WT ASL in lentiviral vectors.
  • Figure 6A is an image of mice at 3 months of age comparing WT with LV GT and shows that the mice were of comparable size and fur pattern.
  • Figure 6B is an image of mice at 6 months comparing AAV GT, WT and untreated Asl Neo/Neo and show that both AAV GT and untreated mice are smaller than WT and have greatly reduced fur pattern.
  • Figure 7A shows that survival and Figure 7B shows that growth of WT and LV Asl Neo/Neo mice were similar to one another, whereas the Untreated Asl Neo/Neo and AAV Asl Neo/Neo both had greatly reduced survival and growth trajectories.
  • FIGS 8A - 8C show that the biomarkers orotic acid (orotate), arginine (citrulline) and argininosuccinic acid in WT and LV Asl Neo/Neo mice were similar to one another at 3 months, whereas the Untreated Asl Neo/Neo and AAV Asl Neo/Neo both showed elevated levels of the biomarkers. These results show that all metabolites typically assessed for ASL function have been normalised in LV Asl Neo/Neo mice but not in AAV Asl Neo/Neo mice.
  • Figures 9A - 9B show that arginine levels in dried blood spots in WT and LV Asl Neo/Neo mice were similar to one another at 3 months, whereas arginine levels in Untreated Asl Neo/Neo and AAV Asl Neo/Neo were similar to one another suggesting a complete correction in LV Asl Neo/Neo but a reversion of phenotype in AAV Asl Neo/Neo after 3 months of treatment.
  • FIGS 10A - 10B show that liver ASL activity was greatly improved by 3 months of age in LV Asl Neo/Neo mice, whereas by 9 months AAV Asl Neo/Neo mice had reverted to similar levels of liver ASL activity as seen in untreated Asl Neo/Neo mice.
  • Example 6 Validation of the safety of the LV.cohASL in murine models and human hepatocytes.
  • Figure 12A shows that both groups exhibited similar growth for the whole timeframe of the experiment. There were no liver tumours detected in either the LV.cohASL- or PBS-treated group.
  • Figure 12B shows that the average liver vector copy number was 0.6 (range 0.1-2) for LV.cohASL-treated mice whereas the vector copy number was undetectable in controls.
  • Figure 12C confirms the high predominance of the vector within the liver and marginal detection in lungs, heart, bone marrow and kidneys.
  • Figure 13A shows the average integration site (IS) per sample was close to 5x10 3 .
  • Figure 13C illustrates the ratio of the frequency of identification for each IS out of the total IS events.
  • the experiments conducted show that using the codon optimised ASL sequence in the lentiviral vector of the present invention, corrects ASLD on a permanent basis in mice and therefore represents a promising treatment for human ASLD patients. Furthermore, it is believed that the therapy of the present invention would not only be suitable for neonatal treatment of ASLD, but also late-onset ASLD due to the corrective nature of the therapy and the regenerative effect of the liver.
  • CMV Cytomegalovirus
  • SEQ ID No. 5 Human immunodeficiency virus type 1 packaging signal (HIV-1 i ) ctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaa ttttgactagcggaggctagaaggagagagatgggtgcgagagcgtc
  • SEQ ID No. 7 Central polypurine tract/central termination seguence (cPPT/CTS) ttttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaact aaagaattacaaaaacaaattacaaaattcaaaattttt
  • SEQ ID No. 12 - Simian virus 40 PolyA signal aacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattcta gttgtggtttgtccaaactcatcaatgtatctta
  • SEQ ID No. 13 Simian virus 40 (SV40) ori atcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgc ctcggcctctgagctattccagaagtagtgaggaggctttttggaggcc

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Abstract

Conformément à la présente invention, l'invention concerne une composition comprenant une séquence d'acide nucléique d'argininosuccinate lyase (ASL) humaine optimisée de SEQ ID No 9 ou une séquence dérivée ayant au moins environ 94 % d'identité de séquence de celle-ci. La présente invention concerne également un vecteur rétroviral d'intégration et/ou lentiviral et/ou gamma incorporant la séquence d'acide nucléique ASL optimisée et son utilisation en tant que thérapie pour traiter une déficience en argininosuccinate lyase (ASLD) ou une maladie ou un état pathologique associé à l'ASLD.
PCT/GB2023/052174 2022-08-19 2023-08-18 Thérapie génique pour le traitement d'une déficience en argininosuccinate lyase Ceased WO2024038287A1 (fr)

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WO2019036484A1 (fr) * 2017-08-15 2019-02-21 The Trustees Of The University Of Pennsylvania Compositions et procédés pour le traitement de l'acidurie argininosuccinique
WO2020104424A1 (fr) * 2018-11-19 2020-05-28 Uniqure Ip B.V. Promoteurs viraux spécifiques du foie et procédés d'utilisation correspondants

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