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WO2025158155A1 - Vecteurs retroviraux - Google Patents

Vecteurs retroviraux

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Publication number
WO2025158155A1
WO2025158155A1 PCT/GB2025/050126 GB2025050126W WO2025158155A1 WO 2025158155 A1 WO2025158155 A1 WO 2025158155A1 GB 2025050126 W GB2025050126 W GB 2025050126W WO 2025158155 A1 WO2025158155 A1 WO 2025158155A1
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Prior art keywords
nucleic acid
pkr
promoter
rna molecule
cell
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Inventor
Matthew TRIDGETT
Sherin Parokkaran JOHNY
Maria ABABI
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Oxford Genetics Ltd
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Oxford Genetics Ltd
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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
    • 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
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • 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
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16051—Methods of production or purification of viral material
    • C12N2740/16052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles

Definitions

  • the present invention relates to processes for the production of retroviral vectors comprising a transgene.
  • the process comprises expressing in a cell: (i) a retroviral vector plasmid comprising a transgene, wherein the transgene is in reverse orientation in the plasmid; (ii) an inhibitor of the transgene RNA; and (iii) an inhibitor of the cell death response.
  • the invention also provides retroviral transfer plasmids and production cell lines for use in the processes of the invention.
  • Retroviruses are positive-sense RNA viruses that undergo a complex life-cycle involving the reverse transcription of their genome into deoxyribonucleic acid (DNA), which subsequently becomes integrated into the host cell genome following viral infection. They are capable of inserting their genomes, as DNA, into almost any loci in the genome of target cells and mediating long-term expression of virus genes, with the DNA being copied into each daughter cell when the infected cell divides. They generate their genome as an un-spliced mRNA molecule by using the cellular RNA polymerase for transcription. The virus genome is then transported into the cytoplasm using a virus protein called Rev.
  • DNA deoxyribonucleic acid
  • the genome is then packaged into virus particles in the cytosol using the virus encoded structural proteins Envelope (Env), Gag and Polymerase (Pol).
  • the retrovirus genome is typically 7-1 Okb in length; in the case of the commonly studied HIV virus, the genome is 9.7kb in length. It exists in each virus particle at 2 copies per virion.
  • retrovirus life cycle affords a number of biotechnological applications, such as the delivery of DNA into the genome of mammalian cells.
  • retroviruses can be modified to contain non-retrovirus glycoproteins in their surface, endowing retrovirus particles with the cellular tropism of the virus from which the glycoprotein originated. This is particularly important when the natural retrovirus glycoprotein has a limited cellular tropism.
  • An example of this is the GP160 glycoprotein of HIV-1 , which has evolved to bind the CD4 receptor and only infects cells bearing this protein on their surface.
  • virus particles are frequently modified to contain a glycoprotein that is different from the natural glycoprotein in a process called pseudotyping. Most commonly, this is achieved with the glycoprotein from vesicular stomatitis virus (VSV-G) to provide a much broader cell tropism.
  • VSV-G vesicular stomatitis virus
  • retroviruses When using retroviruses in the laboratory as tools, they are typically modified to form replication-incompetent vectors that can express either one or more transgenes or shRNA molecules, and these modified viruses provide versatile vectors for cellular transgene expression and engineering.
  • the flexibility of the retrovirus packaging process also allows for varying genome sizes to be accommodated: genomes as small as 3kb and as large as 10-13kb can be packaged, although virus titre can be compromised at these extremes.
  • Retro/lentiviruses are also finding important applications in the field of adoptive cell transfer, most notably to allow expression of hybrid ‘chimeric antigen receptors’ (CAR) within T-cells before cell expansion and reinfusion into patients.
  • CAR hybrid ‘chimeric antigen receptors’
  • the CARs generally have an extracellular antibody structure, and an intracellular structure based on the T- cell receptor, but modified (in 2nd and 3rd generation CARs) to improve the quality of cell stimulation following binding of the outside portion to its antigen.
  • the infection of these cells is only made possible by coating, or pseudotyping, the virus with a broad-tropism glycoprotein, most commonly the VSV-G surface glycoprotein.
  • This protein enables the infection of cells from almost all organs and across many species, including but not limited to, humans, mice, rats, hamsters, monkeys, rabbits, donkeys and horses, sheep, cows and old world apes.
  • retro/lentivirus vectors used for transgene and shRNA expression are typically disabled in a range of ways to remove their ability to replicate and cause disease. This means that in order to grow a batch of infectious virus particles which are capable of a single infection round, for experimental or clinical use, it is necessary to provide several virus genes (and thereby virus proteins) that have been genetically removed from the virus genome at the same time into the cells used for virus packaging. These genes are generally provided in three or four separate plasmids, and co-transfected into cells.
  • the central component is a plasmid encoding the virus vector genome (including any transgenes and associated promoters to regulate transcription in target cells) containing packaging signals to direct the assembling virus particles to incorporate the corresponding RNA into the new virus particles.
  • the genes for other virus proteins such as Gag-Pol, Tat and Rev are generally provided from other plasmids that are co-transfected; and yet another plasmid provides the glycoprotein to be incorporated into the envelope of newly-formed virus particles that will direct their infectious tropism.
  • the gag-pol expression cassette encodes virus capsid and internal structural proteins and polymerase and protease activity.
  • the rev gene acts to enhance nuclear export of retro/lentivirus genomes by binding to a specific region of the virus genome called the Rev Response Element (RRE).
  • RRE Rev Response Element
  • the complexity of retrovirus and lenti virus packaging systems has resulted in a number of ‘generations’ of systems, each with increasing safety on the previous system.
  • three plasmids were used: one plasmid encoding all of the HIV genes except for the envelope gene; a second plasmid to provide a surface glycoprotein (most often VSV-G); and a plasmid containing the virus genome to be packaged.
  • This system has the disadvantage that the plasmid containing the virus genes contained large regions of DNA with homology to the virus genome plasmid, potentially allowing for recombination between plasmids. This could result in infectious virus being produced capable of causing disease.
  • Other problems included the presence of many virus genes that were not needed for the virus production, including VPU, VIF, VPR and Nef.
  • the ‘2nd generation’ systems Five of the nine HIV-1 gene coding regions were removed from the system. This method also resulted in a three-plasmid system, with one plasmid containing the gag-pol genes and the ancillary genes for Tat and Rev proteins, a second plasmid encoding a glycoprotein (most often VSV-G) and a third plasmid that encoded the virus genome to be packaged.
  • the virus genomes in this system typically contain wild-type 5’ Long Terminal Repeats (LTRs) and hence require the tat gene for transcriptional activation and genome production.
  • LTRs Long Terminal Repeats
  • the ‘3rd generation’ system offers a number of advantages (primarily by increasing the number of recombination events required to form replication-competent virus).
  • the ‘3rd generation’ systems also have another significant advantage because they have a modified 5-LTR that includes a promoter, and hence transcription of the genome is not dependent on transcriptional activation by the Tat protein - thereby removing the need for Tat to be encoded in the system. They do not contain the Tat protein on any of the plasmids used.
  • the rev gene was also placed on an individual plasmid. Therefore, in 3rd generation systems, the four plasmids contain 1 : gag-pol, 2: a glycoprotein (most frequently VSV-G), 3: rev, and 4: a plasmid encoding a self-inactivating lentivirus genome containing the transgene or RNA of interest.
  • VSV-G Vesicular Stomatitis Virus
  • the GOI product If the GOI product is membrane-bound, it can be displayed on the surface of the LVV particles. This could alter the physicochemical properties of the LVV particles, thus altering downstream process requirements. Thus, for every new membrane-bound GOI product encoded, a new process must be developed. Silencing the GOI would result in LVV particles that do not display the GOI product on their surface. Thus, changing the GOI would not result in alterations to the physicochemical properties of the LVV, thus no downstream process development would be required, resulting in significant time/cost savings.
  • RNA viral RNA or “vRNA”.
  • vRNA viral RNA
  • the vRNA must not be damaged and its generation by transcription must not be inhibited.
  • targeting the GOI mRNA by RNA interference would also target the vRNA; and preventing transcription of the GOI mRNA would also impose a block on vRNA transcription.
  • the solution provided by the inventors involves reversing the sense direction of the GOI with respect to the orientation of the vRNA in the LVV.
  • the GOI mRNA is no longer encoded within the vRNA, rather the reverse complement of it is.
  • targeting the GOI mRNA by RNA interference does not result in cleavage of the vRNA.
  • the inventors also found that reversing the sense direction of the GOI results in the generation of a large double-stranded RNA (i.e. a double stranded RNA molecule formed by the binding of the vRNA to the GOI mRNA) can trigger cell death in production cells.
  • this process also requires knockdown of the protein kinase R gene (PKR), which is the key initiator of cell death under such circumstances.
  • PKA protein kinase R gene
  • the invention provides retroviral transfer vector plasmids and production cell lines for use in the process of the invention.
  • the invention provides a process for producing a retroviral vector comprising a transgene, the process comprising the steps:
  • transgene capable of expressing a first RNA molecule, optionally wherein the transgene is operably-associated with a second promoter;
  • a retroviral 3’ LTR in the above 5’-3’ order; wherein (iii) is in reverse orientation with respect to the first promoter; (b) a second nucleic acid molecule coding for a second RNA molecule capable of binding to all or part of the first RNA molecule;
  • the retroviral vector is a lentiviral vector.
  • the invention provides a retroviral transfer plasmid comprising:
  • transgene capable of expressing a first RNA molecule, optionally wherein the transgene is operably-associated with a second promoter; and (iv) a retroviral 3’ LTR; in the above 5’-3’ order; wherein (iii) is in reverse orientation with respect to the first promoter;
  • a second nucleic acid molecule coding for a second RNA molecule capable of binding to all or part of the first RNA molecule
  • a third nucleic acid molecule coding for a PKR inhibitor preferably coding for an RNA molecule capable of binding to all or part of a PKR RNA molecule, more preferably wherein the PKR inhibitor is an anti-PKR shRNA.
  • the invention also provides a kit comprising:
  • transgene capable of expressing a first RNA molecule, optionally wherein the transgene is operably-associated with a second promoter; and (iv) a retroviral 3’ LTR; in the above 5’-3’ order; wherein (iii) is in reverse orientation with respect to the first promoter;
  • a third nucleic acid molecule coding for a PKR inhibitor preferably coding for an RNA molecule capable of binding to all or part of a PKR RNA molecule, more preferably wherein the PKR inhibitor is an anti-PKR shRNA; optionally wherein the first, second and third nucleic acid molecules are present on one or more plasmids, and wherein:
  • the second and third nucleic acid molecules are located on the same plasmid;
  • the first, second and third nucleic acid molecules are located on the same plasmid.
  • the invention also provides a host cell comprising: a) a first nucleic acid molecule comprising:
  • transgene capable of expressing a first RNA molecule, optionally wherein the transgene is operably-associated with a second promoter;
  • a third nucleic acid molecule coding for a PKR inhibitor preferably coding for an RNA molecule capable of binding to all or part of a PKR RNA molecule, more preferably wherein the PKR inhibitor is an anti-PKR shRNA.
  • Lentiviruses are a subset of the retroviridae family that are increasingly being vectorised and then used for transgene delivery and protein expression, particularly in progenitor cell populations such as haematopoietic stem cells and T cells. Unlike most retroviruses, lentiviruses are able to deliver their genome, or modified forms thereof, independent of the cell cycle, and often achieve higher efficiency of cellular infection in a shorter time frame. This makes them a much more effective viral vector for both research and clinical use.
  • the lentivirus family consists of 10 viruses at present. These species are divided into five groups including: Bovine lentivirus group (Bovine immunodeficiency virus and Jembrana disease virus), Equine lentivirus group (Equine infectious anaemia virus, Feline lentivirus group, Feline immunodeficiency virus, Puma lentivirus), Ovine/caprine lentivirus group (Caprine arthritis encephalitis virus, Visna/maedi virus), Primate lentivirus group, (Human immunodeficiency virus 1 , Human immunodeficiency virus 2, Simian immunodeficiency virus).
  • the lentivirus is Human immunodeficiency virus 1 , Simian immunodeficiency virus or Equine infectious anaemia virus.
  • the lentivirus is Human immunodeficiency virus 1 or Equine infectious anaemia virus.
  • retroviral vector refers to a non-replicative retrovirus-like particle that is capable of transferring genetic material from the cell in which it was produced to a target cell.
  • retroviral vectors include gamma-retroviral vectors (e.g. vectors derived from murine leukaemia viruses) and lentiviral vectors.
  • the retroviral vector is a lentiviral vector.
  • Retroviral vectors have RNA, preferably single-stranded RNA, genomes. Therefore, the retroviral vectors produced by the process of the invention have RNA, preferably singlestranded RNA, genomes.
  • the following elements are commonly present in the genomes of lentiviral vectors, but are not all essential for the invention: packaging signal, Rev response element, central poly-purine tract, chain termination sequence, primer activation sequence, major splice donor site and woodchuck hepatitis virus post-transcriptional regulatory element.
  • the retroviral vectors of the invention may have one or more or all of the aforementioned elements.
  • retroviral vector genome refers to elements (ii), (iii) and (iv).
  • retroviral vector plasmid refers to a plasmid that is useful in the production of retroviral vectors.
  • the term “lentiviral vector plasmid” refers to a vector or plasmid which is useful in the production of lentiviral vector.
  • the retroviral vector plasmid may be a transfer plasmid, packaging plasmid, an envelope plasmid or a packaging/envelope plasmid.
  • the lentiviral vector plasmid may be a transfer plasmid, packaging plasmid, an envelope plasmid or a packaging/envelope plasmid.
  • the retroviral vector plasmid is a transfer plasmid.
  • a transfer plasmid is a plasmid which encodes the retroviral vector genome.
  • the cells are preferably mammalian cells.
  • mammalian cells include those from any organ or tissue from humans, mice, rats, hamsters, monkeys, rabbits, donkeys, horses, sheep, cows and apes.
  • the cells are human cells.
  • the cells may be primary or immortalised cells.
  • the cells may be adherent, or suspension adapted.
  • the cells may form a clonal or heterogeneous population.
  • Preferred cells include HEK-293, HEK 293T, HEK-293E, HEK-293 FT, HEK-293S, HEK-293SG, HEK-293 FTM, HEK-293SGGD, HEK-293A, MDCK, C127, A549, HeLa, CHO, mouse myeloma, PerC6, 911 , and Vero cell lines.
  • HEK-293 cells have been modified to contain the E1A and E1 B proteins and this allows the creation of viruses that have a deletion of the E1 A and E1 B regions to be grown in this cell line by trans-complementation.
  • PerC6 and 911 cells contain a similar modification and can also be used.
  • the human cells are HEK293, HEK293T, HEK293A, PerC6 or 911 .
  • Other preferred cells include CHO and VERO cells.
  • the cells of the invention are capable of inducibly-expressing the env and gag-pol genes.
  • the cells may be isolated cells, e.g. they are not situated in a living animal.
  • the cells are recombinant cells, i.e. they are not naturally-occurring.
  • the first nucleic acid molecule is preferably a DNA molecule, more preferably a doublestranded DNA molecule.
  • the first nucleic acid may be termed a “retroviral vector plasmid” or “transfer plasmid”.
  • the first nucleic acid molecule is a plasmid or vector, located episomally within the cell.
  • the first nucleic acid may be located within a bacterial artificial chromosome or a yeast artificial chromosome.
  • the first nucleic acid molecule is integrated into the genome of the cell.
  • the first promoter is one which is capable of driving transcription of the retroviral vector genome, i.e. elements (ii), (iii) and (iv).
  • the first promoter is a cytomegalovirus (CMV) promoter or a Rous sarcoma virus (RSV) promoter.
  • CMV cytomegalovirus
  • RSV Rous sarcoma virus
  • the first promoter is a cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • the first promoter is operably-associated with the retroviral vector genome (i.e. elements (ii), (iii) and (iv).
  • Retroviral long terminal repeats are found on either side of a retroviral provirus.
  • the 5’ LTR acts as an RNA pol II promoter.
  • Wild-type retroviral LTRs comprise U3-R-U5 regions.
  • the U3 (Unique 3’) region contains sequences necessary for activation of viral genomic RNA transcription.
  • Tat binds to the R (Repeat) region. Transcription begins, by definition, at the beginning of R and proceeds through U5 and the rest of the provirus. The U5 (Unique 5') is transcribed to form the 5' end of the viral genomic RNA.
  • Third generation retroviral vectors use a hybrid 5' LTR with a constitutive promoter, such as CMV or RSV.
  • the second promoter which is optional, is one which is capable of driving transcription of the transgene.
  • the transgene is transcribed from the second promoter in a sense orientation (with respect to the second promoter; antisense with respect to the first promoter). In the absence of a second promoter, expression of the transgene may be driven from a viral transcript promoter.
  • suitable second promoters include the Spleen focus-forming virus (SFFV) promoter, elongation factor 1 -alpha 1 (EF-1a) promoter, elongation factor 1 -alpha short (EFS) promoter, Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, U6 promoter and H1 promoter. Further examples include pol ll/pol III promoters.
  • the second promoter is selected from the group consisting of the Spleen focus-forming virus (SFFV) promoter, elongation factor 1 -alpha 1 (EF-1a) promoter and elongation factor 1 -alpha short (EFS) promoter.
  • the second promoter is the SFFV promoter.
  • the second promoter when present, is operably-associated with the transgene.
  • operably-associated refers to the association of nucleic acid molecules on a single nucleic acid fragment so that the function of one nucleic acid molecule affects the function of the other nucleic acid molecule.
  • the nucleic acid molecules may be juxtaposed, adjacent or contiguously-linked; one may be upstream of the other.
  • the terms imply a physical connection between the nucleic acid molecules within a distance which allows the function of one nucleic acid molecule to affect the function of the other nucleic acid molecule.
  • a promoter is operably-associated with a nucleic acid molecule when the promoter is capable of affecting (i.e.
  • Coding polynucleotides in sense or antisense orientation can be operably- associated with regulatory polynucleotides.
  • transgene and “gene of interest” (GOI) are used interchangeably herein.
  • the transgene is capable of expressing, i.e. it encodes, a first RNA molecule.
  • the transgene will generally comprise a 5’ untranslated region, a polypeptide-encoding region or a functional RNA-encoding region, and a 3’ untranslated region.
  • the first RNA molecule may be a functional RNA molecule or it may encode a polypeptide.
  • functional RNA molecules include shRNA and miRNA.
  • the transgene is one whose expression within a mammalian cell would be toxic to that cell.
  • the transgene codes for a therapeutic polypeptide or a fragment thereof. In some embodiments, the transgene encodes one or more polypeptides.
  • therapeutic polypeptides examples include antibodies, CAR-T molecules, scFV, BiTEs, DARPins and T-cell receptors.
  • the therapeutic polypeptide is a G-protein coupled receptor (GPCR), e.g. DRD1.
  • GPCR G-protein coupled receptor
  • the therapeutic polypeptide is a functioning copy of a gene involved in human vision or retinal function, e.g. RPE65 or REP.
  • the therapeutic polypeptide is a functioning copy of a gene involved in human blood production or is a blood component, e.g. Factor IX, or those involved in beta and alpha thalassemia or sickle cell anaemia.
  • the therapeutic polypeptide is a functioning copy of a gene involved in immune function such as that in severe combined immune-deficiency (SCID) or Adenosine deaminase deficiency (ADA-SCID).
  • SCID severe combined immune-deficiency
  • ADA-SCID Adenosine deaminase deficiency
  • the therapeutic polypeptide is a protein which increases/decreases proliferation of cells, e.g. a growth factor receptor.
  • the therapeutic polypeptide is an ion channel polypeptide.
  • the therapeutic polypeptide is an immune checkpoint molecule.
  • the immune checkpoint molecule is PD1 , PDL1 , CTLA4, Lag1 or GITR.
  • the transgene encodes a CRISPR enzyme (e.g. Cas9, dCas9, Cpf1 or a variant or derivative thereof) or a CRISPR sgRNA.
  • a CRISPR enzyme e.g. Cas9, dCas9, Cpf1 or a variant or derivative thereof
  • a CRISPR sgRNA e.g. Cas9, dCas9, Cpf1 or a variant or derivative thereof
  • transgene (GOI) gene products are known to be either directly toxic to mammalian cells or to interfere with lenti viral vector production:
  • BAX B-cell lymphoma 2-associated X-protein
  • the effects could include one or more of the following: Slow growth of production cells compared to the cells from which they were derived; an unacceptable level of cell death during LVV production; low LVV production compared to production cells that do not encode a toxic GOI; and reduced transduction efficiency of LVV that is produced, e.g. coagulation factor VIII is known to impact the display of VSV-G (vesicular stomatitis virus G protein) on the surface of LVV particles, which reduces infectious titre without impacting physical titre (Radcliffe et al., Gene Then, 2008 Feb; 15: 289-297). Silencing a toxic GOI during LVV production would thus be expected to ameliorate these issues.
  • the transgene encodes one of the above- mentioned gene products.
  • CARs chimeric antigen receptors
  • transgene e.g. anti-CD19 CAR and anti-B-cell maturation antigen CAR
  • they would also be expected to be displayed on the lentiviral vector surface, as it is an enveloped viral vector whose envelope is derived from the production cell membrane.
  • silencing CAR genes during lentiviral vector production would be beneficial as it would prevent the display of the transgene gene product on the lentiviral vector surface, making the end-product consistent, making downstream processing simpler and reducing the risk of immunogenicity.
  • the maximum cloning capacity of a lentiviral vector is about 8.5 kb, but inserts of greater than about 3 kb are packaged less efficiently.
  • the transgene size is less than 3 kb.
  • the 3’ LTR also comprises U3-R-U5 regions.
  • the transcription which started in the 5' LTR terminates in the 3’ LTR by the addition of a poly A tract just after the R sequence.
  • the 3’ LTR includes a deletion relative to the wild-type 3’ LTR, rendering the retroviral vector “self-inactivating” (SIN) after integration into a mammalian genome.
  • the second nucleic acid molecule codes for (and is capable of expressing) a second RNA molecule which is capable of binding to all or part of the first RNA molecule.
  • the second nucleic acid molecule may comprise a third promoter, operably- associated with a nucleotide sequence encoding the second RNA molecule.
  • the third promoter may be a constitutive or inducible promoter.
  • the function of the second RNA molecule is to bind to all or part of the first RNA molecule in order to inhibit or prevent the translation of the first RNA molecule in the cell. If the first RNA molecule encodes a functional RNA, then the function of the second RNA molecule is to bind to all or part of the first RNA molecule in order to inhibit or prevent the activity of that functional RNA.
  • the first and second RNA molecules will bind to each other by Watson-Crick binding.
  • the second RNA molecule will have a ribonucleotide sequence which is antisense compared to all or part of the ribonucleotide sequence of the first RNA molecule.
  • ribonucleotide sequences of the first and second RNA molecules will therefore be fully or partially complementary.
  • the first and second RNA molecules have at least 50%, 60%, 70%, 80% or 90% complementary sequence identity over all or part of their lengths.
  • the ribonucleotide sequences of the first and second RNA molecules are complementary or partially complementary over a stretch of at least 30 bp.
  • shRNA molecules include short hairpin RNA (shRNA), short interfering RNA (siRNA), microRNA (miRNA) and primary microRNA (pri-miRNA), having a ribonucleotide sequence which is complementary to all or part of the first RNA molecule.
  • shRNA molecules comprise a sense strand, stem loop and an antisense strand.
  • the sense strand and antisense strand may be 17-22 or 19-22 nucleotides each, and the loop may be 4-11 nucleotides.
  • the sense strand and antisense strand are about 21 nucleotides each, and the loop is 7 nucleotides.
  • a siRNA may, for example, be 20-25 nucleotides in length.
  • the second RNA molecule is a miRNA which binds to a complementary sequence in the 5’ UTR or 3’UTR (preferably the 3’UTR) of the first RNA molecule.
  • the complementary sequence in the 5’ UTR or 3’UTR of the first RNA molecule may be a sequence which is part of the endogenous transgene sequence or the complementary sequence in the 5’ UTR or 3’UTR may be a heterologous sequence (i.e. designed for binding to the miRNA).
  • the second RNA molecule is a shRNA which comprises a sense strand, stem loop and an antisense strand, wherein the base-pairing between the sense and antisense strands at the 5’-end of the antisense strand (e.g. within 1-5 nucleotides of the stem loop) is destabilised (e.g. by the presence of 1 , 2, 3, 4 or 5 non- complementary nucleotides). This enhances the strand specificity of the shRNA, i.e. it will be less prone to aberrantly targeting the viral RNA.
  • the second RNA molecule is an miRNA (micro RNA) wherein the nucleotide sequence of the miRNA is partially or fully complementary to the nucleotide sequence of the first RNA molecule except for 1 , 2 or 3 nucleotides thus preventing the cleavage of the first RNA molecule.
  • miRNA miRNA
  • the rationale is that due to the partial mismatch, mRNA cleavage is prevented while retaining translational repression by routes that do not require mRNA cleavage. Thus any aberrant targeting of the viral RNA will not disrupt packaging of the viral RNA into the viral vector.
  • the second RNA molecule is a primary miRNA (pri-miRNA).
  • pri-miRNA primary miRNA
  • the antisense strand is loaded into a nucleoprotein complex which performs gene silencing.
  • the antisense strand is preferentially loaded, it is also possible for the sense strand to be loaded. This means that a fraction of the shRNA-derived gene silencing complexes in the cell will target the opposite strand to that which is intended to be targeted; in this case, this would be the viral RNA. Conversion of the shRNA to a pri- miRNA as per Kaadt et al. (Molecular Therapy Nucleic Acids, 2018 Nov; 14: 318-328) abolishes the aberrant loading of the sense strand. Thus using this approach, one can be certain that there would be no targeting of the viral RNA by the silencing system.
  • a second advantage is that conversion of shRNA to pri-miRNA as per Kaadt et al. would permit driving the expression of the second RNA molecule by any Pol II promoter, which could be much stronger than the Pol III promoters that must be used with shRNAs. This could enable an increase in the amount of second RNA molecule in the production cells, thus achieving more effective GOI knockdown.
  • the second RNA molecule could be designed to target the 5’ or 3’ UTR, or a custom sequence that is inserted into the 5’ or 3’ UTR, of the GOI (transgene).
  • the second nucleic acid molecule is expressed (to produce the second RNA molecule) in the cell in which the first nucleic acid molecule is expressed.
  • the second nucleic acid molecule may, for example, be integrated into the (nuclear or mitochondrial) genome of the cell or it may be present episomally, e.g. on a plasmid or vector within the cell.
  • the second nucleic acid may be located within a bacterial artificial chromosome or a yeast artificial chromosome.
  • One or more further nucleic acid molecules expressing retroviral gag-pol, env and rev genes are also present within the cell. These genes (and the polypeptides that they encode) are required in order to produce and package retroviral vectors within the cell.
  • the env, gag-pol and rev genes are preferably viral genes or derived from viral genes. More preferably, they are retroviral genes or derived from retroviral genes.
  • retroviruses examples include lentiviruses, alpha-retroviruses, gammaretroviruses (e.g. murine leukaemia viruses) and foamy-retrovi ruses.
  • retrovirus is a lentivirus.
  • the env, gag, pol and rev genes may be from one or more different viruses (e.g. 2, 3 or 4 different viruses).
  • the env gene may be from Rhabdoviridae (e.g. VSV- G) whilst other the gag, pol and rev genes may be from HIV-1 .
  • env is a gene that encodes the protein which forms the viral envelope.
  • the expression of the env gene enables retroviruses to target and attach to specific cell types, and to infiltrate the target cell membrane. Examples of the env gene include the HIV-1 env gene and derivatives thereof.
  • the env gene codes for the gp160 protein which forms a homotrimer, and is cleaved into gp120 and gp41 by the host cell protease, Furin.
  • the HIV-1 env nucleotide and amino acid sequences are given in SEQ ID NOs: 1 and 2, respectively.
  • HIV-1 env gene refers preferably to a nucleotide sequence having the sequence given in SEQ ID NO: 1 or a nucleotide sequence encoding SEQ ID NO: 2, or a nucleotide sequence having at least 80%, 85% 90%, 95% or 99% sequence identity thereto and which encodes a gp160 protein which is capable of forming a homotrimer and is capable of being cleaved into gp120 and gp41 polypeptide by the HIV-1 protease.
  • the viral envelope may be pseudo-typed by using an env gene from a virus such as Vesicular Stomatitis virus (VSV), e.g. the VGV-G gene, or a derivative thereof.
  • VSV Vesicular Stomatitis virus
  • the VSV-G protein is a single-pass membrane glycoprotein. It mediates a broad infectious tropism.
  • the gene is encoded by a 1536 bp open reading frame and produces a protein consisting of 511 amino acids.
  • the protein contains a 16 amino signal peptide at the N-terminus (amino acid sequence: MLSYLIFALAVSPILG, SEQ ID NO: 11) which is cleaved from the mature protein during export through the secretory pathway to the cell surface.
  • the glycoprotein contains an extracellular region of 458 amino acids and a membrane spanning region (transmembrane region) of 21 amino acids followed by an intracellular (cytosolic) C-terminal region of 22 amino acids.
  • VSV-G protein The shuttling of VSV-G protein from the endoplasmic reticulum is rapid, and this is achieved by the specific trafficking signals in the C-terminal tail, including a DxE motif (where x is any amino acid) within the broader trafficking signal Tyr-Thr-Asp-lle-Glu-Met that contains the DxE motif (Sevier et al., Mol. Biol. Cell. 2000 Jan; 11 (1): 13-22).
  • the efficiency of export of VSV-G protein may in part contribute to its effectiveness for retrovirus and lentivirus production.
  • VSV-G receptor is frequently described as a non-specific fusogenic protein; however, it was recently determined the VSV-G binds to the low-density lipid receptor (LDL-R) (Finkelstein et al., Proc. Natl. Acad. Sci. USA 2013; 110(18)7306-7311), which explains its broad cellular tropism and broad application in retrovirus and lentivirus pseudo-typing.
  • LDL-R low-density lipid receptor
  • VSV-G gene refers preferably to a nucleotide sequence having the sequence given in SEQ ID NO: 3 or a nucleotide sequence encoding SEQ ID NO: 4, or a nucleotide sequence having at least 80%, 85% 90%, 95% or 99% sequence identity thereto and which encodes a polypeptide which is capable of attaching to the LDL receptor.
  • gag-pol includes contiguous/overlapping gag-pol genes and independent gag and pel genes.
  • the Gag-Pol protein of lentiviruses is produced as a single poly-protein that encodes a protease that enables the proteolytic cleavage of the Gag-Pol protein into a number of smaller proteins serving a number of virus functions.
  • the HIV-1 Gag protein is produced from the first translated open reading frame from the 5’-end of the virus genome and contains a sequence known as the frame-shift sequence. This signal causes the translating ribosome to shift back on the mRNA molecule one base during translation approximately every 1 in 20 translation runs. This process produces the Gag-Pol protein.
  • the result is that lentivirus produce Gag and Gag-Pol at an approximate ratio of 1 :20.
  • the Gag protein encodes three major structural proteins: p18, p24 and p15.
  • the Pol protein segment also encodes three major proteins called p10 (protease), p66/55 (reverse transcriptase) and p32 (integrase).
  • the protease is responsible for all of the cleavage events required to produce each of these proteins by proteolytic cleavage.
  • the protease recognition sequences that define these cleavage events are poorly defined, suggesting that the protease has broad specificity. This is therefore likely to result in the cleavage of proteins that are not virus related.
  • the expression of Gag-Pol proteins is reported to be highly toxic to cells because of this (Blanco et al., The Journal of Biochemistry, 278, 2, 1086-1093, 2003).
  • the coding sequences of the gag and pol genes overlap.
  • the coding sequences of the gag and pol genes of the invention may be contiguous, noncontiguous, overlapping or non-overlapping.
  • the gag-pol sequence is from a lenti virus.
  • the gag, pol and gag- pol genes include HIV-1 gag-pol genes and derivatives thereof.
  • the gag-pol genes are from HIV-1 .
  • the reading frames of the gag and pol genes overlap, i.e. in a gag-pol gene.
  • the HIV-1 gag-pol nucleotide sequence is given in SEQ ID NO: 5.
  • HIV-1 gag-pol gene refers preferably to a nucleotide sequence having the sequence given in SEQ ID NO: 5, or a nucleotide sequence having at least 80%, 85% 90%, 95% or 99% sequence identity thereto and which encodes matrix, capsid and nucleocapsid proteins, and a reverse transcriptase, integrase, and protease.
  • Rev is a trans-activating protein that is essential to the regulation of HIV-1 protein expression.
  • a nuclear localization signal is encoded in the rev gene, which allows the Rev protein to be localized to the nucleus, where it is involved in the export of unspliced and incompletely spliced mRNAs.
  • Rev binds to a region in the lentivirus genome called the Rev Response Element which allows the nuclear export of unspliced, full length genomes, which is essential for lentivirus production.
  • rev gene examples include the HIV-1 rev gene and derivatives thereof.
  • the HIV- 1 rev nucleotide and Rev amino acid sequences are given in SEQ ID NOs: 6 and 7, respectively.
  • HIV-1 rev gene refers preferably to a nucleotide sequence having the sequence given in SEQ ID NO: 6 or a nucleotide sequence encoding SEQ ID NO: 7, or a nucleotide sequence having at least 80%, 85% 90%, 95% or 99% sequence identity thereto and which encodes a protein which is capable of binding to the Rev Response Element (RRE).
  • RRE Rev Response Element
  • the further nucleic acid molecules do not comprise a nucleic acid comprising a rev gene.
  • VSV-G is generally cytotoxic to cells. It is capable of inducing cell fusion and the formation of syncytia. Some of the gag-pol gene products are also cytotoxic. In particular, the pel gene encodes a protease that cleaves proteins within the cell and leads to cell death.
  • the expression of one or more apoptosis inhibitors mitigates or prevents apoptosis of the cell which would otherwise have been initiated by the cytotoxicity of the cytotoxic polypeptide(s). Therefore, the further nucleic acid molecules of the invention may additionally comprise one or more nucleotide sequences encoding apoptosis inhibitors.
  • the one or more apoptosis inhibitors may independently, for example, be polypeptide or RNA.
  • the further nucleic acid molecules of the invention additionally comprise 1 , 2, 3, 4 or 5, more preferably, 1 or 2 nucleotide sequences encoding apoptosis inhibitors.
  • the apoptosis inhibitor is an inhibitor of the APAF-1 (e.g. AVEN), Caspase 9 (e.g. IAP or XlAP), BAK, BAX, BOK or BAD (e.g. BCL2, E1 B-19K or BCL-XL) pathway.
  • APAF-1 e.g. AVEN
  • Caspase 9 e.g. IAP or XlAP
  • BAK e.g. IAP or XlAP
  • BAX e.g. BOK or BAD
  • BCL2 e.g. BCL2, E1 B-19K or BCL-XL
  • more than one gene is used that inhibits more than one apoptosis pathway or step (e.g. AVEN combined with E1 B-19K) to provide improved
  • the one or more of the apoptosis inhibitor is one which inhibits an apoptotic protein whose production is stimulated by loss of cell membrane integrity, by cell-cell fusion or by syncytia formation or one which is stimulated by a protease that cleaves proteins within the cell.
  • Examples of apoptosis-inhibiting polypeptides include Celovirus GAM1 , Adenovirus E4 Orf6, Adenovirus E1 B 55K, Adenovirus E1 B 19K, Myxoma virus M11 L, Cytomegalovirus IE1 , Cytomegalovirus IE2, Baculovirus p35, Baculovirus IAP-1 , Herpesvirus US3, Herpesvirus Saimiri ORF16, Herpes Simplex 2 LAT ORF 1 , Human XIAP, African Swine Fever ASFV-5-HL (LMW-5-HL/A179L), Kaposi’s Sarcoma virus KSbcl2, Vaccinia virus SPI-2, Cowpoxvirus CrmA, Epstein Barr virus BHRF1 , Epstein Barr virus EBNA-5, Epstein Barr virus BZLF-1 , Papillomavirus E6, Human Aven, Human BCL2 and Human BCL-XL.
  • one or more of the apoptosis inhibitors is an RNA, preferably an antisense or shRNA.
  • RNA apoptosis inhibitors include Herpesvirus LAT and Adenovirus VA1 .
  • the apoptosis inhibitors are selected from the group consisting of IAP1 , EBNA5 and BCL-XL.
  • Particularly-preferred combinations of apoptosis inhibitors include: IAP1 + EBNA5; IAP1 + BCL-XL; and EBNA5 + BCL-XL.
  • Nucleotide sequences of apoptosis inhibitors IAP1 , EBNA5 and BCL-XL are given herein as SEQ ID NOs: 8, 9 and 10, respectively.
  • nucleic acid molecules of the invention which additionally comprises a nucleotide sequence encoding an apoptosis inhibitor comprising SEQ ID NO: 8, 9 or 10, or a nucleotide sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto.
  • Each of the genes in the further nucleic acid molecules are preferably operably- associated with one or more regulatory elements. This ensures that the appropriate polypeptide is expressed at the desired level and at the desired time.
  • regulatory elements includes one or more of an enhancer, promoter, intron, polyA, insulator or terminator.
  • genes used in the vectors herein are preferably separated by polyA signals and/or insulators in an effort to keep transcriptional read-through to other genes to a minimum and also to insulate the genes which it is desired to repress (VSV-G and gag-pol) under normal circumstances from genes which it is desired to be expressed (e.g. TetR and the apoptosis inhibitors).
  • the env and gag-pol genes are operably associated with different regulatory elements, e.g. different promoter, different intron, different polyA, different insulator and/or different terminator sequences.
  • the further nucleic acid molecules may, for example, independently be integrated into the (nuclear or mitochondrial) genome of the cell or they may be present episomally, e.g. on a plasmid or vector within the cell.
  • One or more of the nucleic acids may be located within a bacterial artificial chromosome or a yeast artificial chromosome within the cell. Preferably, they are all integrated into the (nuclear or mitochondrial) genome of the cell.
  • the binding of the first RNA molecule to the retroviral vector RNA molecule (partially or completely) generates a double-stranded RNA molecule which can trigger a cell-death response within the cell, thus leading to the death of the cell.
  • RNA molecules which can trigger a cell-death response within the cell, thus leading to the death of the cell.
  • Mammalian cells perceive the presence of double-stranded RNA molecules as being associated with viral infection. It is therefore necessary to prevent the triggering of this cell-death response within the cell.
  • the cell death response is initiated by the activation of the endogenous protein kinase R.
  • the cell’s nuclear genome comprises a PKR gene which encodes a PKR RNA, which encodes a PKR polypeptide.
  • PPKR Protein kinase R
  • EIF2AK2 eukaryotic translation initiation factor 2-alpha kinase 2
  • PKR is a serine/tyrosine kinase that is 551 amino acids long.
  • the cell death response may be inhibited or prevented by expressing an inhibitor of the endogenous PKR gene or endogenous PKR polypeptide within the cell.
  • the PKR inhibitor may be expressed in the cell either constitutively or inducibly.
  • the cell death response is prevented or inhibited within the cell by expressing in the cell: (d) a third nucleic acid molecule coding for a PKR inhibitor.
  • the third nucleic acid molecule may be present within the cell genome or may be present episomally within the cell.
  • the third nucleic acid may located within a bacterial artificial chromosome or a yeast artificial chromosome within the cell.
  • the PKR inhibitor is present in the cell media surrounding the cell.
  • the PKR inhibitor is a RNA molecule having a ribonucleotide sequence which is complementary to all or part of the endogenous PKR RNA.
  • PKR inhibitors include short hairpin RNA (shRNA), short interfering RNA (siRNA), microRNA (miRNA) and primary microRNA (pri-miRNA) having a ribonucleotide sequence which is complementary to all or part of the PKR RNA.
  • shRNA short hairpin RNA
  • siRNA short interfering RNA
  • miRNA microRNA
  • pri-miRNA primary microRNA having a ribonucleotide sequence which is complementary to all or part of the PKR RNA.
  • the PKR inhibitor is a shRNA against the PKR RNA.
  • the PKR inhibitor is an inhibitor of the PKR polypeptide.
  • inhibitors of the PKR polypeptide include C16 (PKRi, GW 506033X; CAS number 608512-97-6); and the small molecule inhibitors described in Jammi et al. (Biochemical and Biophysical Research Communications, vol. 308, Issue 1 , 15 August 2003, Pages 50-57) and Cusack et al. (Bioorganic & Medicinal Chemistry Letters Volume 79, 1 January 2023, 129047).
  • the PKR inhibitor is a shRNA which comprises a sense strand, stem loop and an antisense strand, wherein the base-pairing between the sense and antisense strands at the 5’-end of the antisense strand (e.g. within 1 -5 nucleotides of the stem loop) is destabilised (e.g. by the presence of 1 , 2, 3, 4 or 5 non-complementary nucleotides).
  • the cell death response is prevented or inhibited by performing a process in a cell wherein the PKR genes have been knocked out, i.e. the PKR genes are not functional or have been fully or partially deleted.
  • the first and second nucleic acid molecules are present on a plasmid together with a third nucleic acid molecule which encodes a PKR inhibitor.
  • a nucleotide (gene) sequence in accordance with this embodiment is as shown in SEQ ID NO: 16.
  • the advantages of this system compared to the system in which the transfer plasmid and a PKR inhibitor plasmid are separate are as follows: the lenti viral vector production process is simplified as only one plasmid needs to be prepared and transfected; and the ratio of anti-PKR RNA to GOI mRNA would be increased compared to the system with separate plasmids, potentially improving the effectiveness of gene silencing.
  • (b) i.e. the second nucleic acid
  • the plasmid additionally comprises:
  • a third nucleic acid molecule coding for a PKR inhibitor preferably wherein the PKR inhibitor is an anti-PKR RNA molecule which is capable of binding to all or part of a PKR RNA molecule, more preferably wherein the PKR inhibitor is an anti-PKR shRNA.
  • a retroviral transfer plasmid comprising: (a) a first nucleic acid molecule comprising:
  • transgene capable of expressing a first RNA molecule, optionally wherein the transgene is operably-associated with a second promoter;
  • a third nucleic acid molecule coding for a PKR inhibitor preferably coding for an RNA molecule capable of binding to all or part of a PKR RNA molecule, more preferably wherein the PKR inhibitor is an anti-PKR shRNA.
  • the invention also provides a process as described herein wherein: a first nucleic acid molecule of the invention; a second nucleic acid molecule of the invention; and a third nucleic acid molecule coding for a PKR inhibitor, preferably wherein the PKR inhibitor is an anti-PKR RNA molecule which is capable of binding to all or part of a PKR RNA molecule, more preferably wherein the PKR inhibitor is an anti- PKR shRNA, are all present in the cell and are located on one or more plasmids; and wherein:
  • the second and third nucleic acid molecules are located on the same plasmid;
  • the invention also provides a host cell comprising: a first nucleic acid molecule of the invention; a second nucleic acid molecule of the invention; and a third nucleic acid molecule coding for a PKR inhibitor, preferably wherein the PKR inhibitor is an anti-PKR RNA molecule which is capable of binding to all or part of a PKR RNA molecule, more preferably wherein the PKR inhibitor is an anti-PKR shRNA.
  • kits comprising: a first nucleic acid molecule of the invention; a second nucleic molecule acid of the invention; and a third nucleic acid molecule coding for a PKR inhibitor, preferably wherein the PKR inhibitor is an anti-PKR RNA molecule which is capable of binding to all or part of a PKR RNA molecule, more preferably wherein the PKR inhibitor is an anti-PKR shRNA; optionally wherein the first, second and third nucleic acid molecules are all present on one or more plasmids, and wherein:
  • the second and third nucleic acid molecules are located on the same plasmid;
  • the first, second and third nucleic acid molecules are located on the same plasmid.
  • the first and second nucleic acid molecules are present on a plasmid together, within the cell. Furthermore, a fragment of a PKR gene is inserted within the 3’ UTR of the transgene; and the ribonucleotide sequence of the second RNA molecule is tailored to be complementary to the fragment of the PKR gene.
  • a nucleotide (gene) sequence in accordance with this embodiment is as shown in SEQ ID NO: 17.
  • the fragment of the PKR gene within the 3’ UTR of the transgene i.e. part of the first RNA molecule
  • the anti-PKR RNA molecule i.e. the second RNA molecule
  • the anti-PKR RNA molecule will also target the cell’s endogenous PKR RNA, thus preventing initiation of the cell death response within the cell.
  • the advantage of this system is as follows: since the sequence targeted by the anti- PKR RNA molecule to silence the transgene expression is outside of the codondetermining sequence (CDS) of the transgene, the transgene could be modified or replaced without the need to screen a panel of anti-transgene shRNAs to find an effective one. This shortens development timelines when customising this system for novel applications.
  • CDS codondetermining sequence
  • the first nucleic acid molecule comprises:
  • transgene capable of expressing a first RNA molecule, optionally wherein the transgene is operably-associated with a second promoter, wherein the transgene (and first RNA molecule) comprises a 5’ or 3’ untranslated region (UTR) which comprises a fragment of a PKR gene; and
  • the second nucleic acid molecule codes for an anti-PKR RNA molecule, wherein the anti-PKR RNA molecule is capable of binding to both the 5’ or 3’ UTR of the first RNA molecule (i.e. to the portion of the first RNA molecule having the ribonucleotide sequence of the fragment of the PKR gene) and endogenous PKR mRNA expressed from the cell’s genome.
  • the anti-PKR RNA molecule is an anti-PKR shRNA or miRNA.
  • a retroviral transfer plasmid comprising:
  • transgene capable of expressing a first RNA molecule, optionally wherein the transgene is operably-associated with a second promoter, wherein the transgene comprises a 5’ or 3’ untranslated region (UTR) which comprises a fragment of a PKR gene; and
  • a second nucleic acid molecule coding for an anti-PKR RNA molecule wherein the anti-PKR RNA molecule is capable of binding to both the 5’ or 3’ UTR of the first RNA molecule (i.e. to the portion of the first RNA molecule having the ribonucleotide sequence of the fragment of the PKR gene) and a PKR mRNA molecule (such as that expressed from an endogenous PKR gene within a cell).
  • the anti-PKR RNA molecule is an anti-PKR shRNA or miRNA.
  • the length of the fragment of the PKR gene may, for example, be 19 to 1000. nucleotides, preferably 19 to 100 nucleotides. In some preferred embodiments, the nucleotide (gene) sequence of the fragment of the PKR gene is:
  • the invention also provides a cell, preferably one as disclosed herein, which comprises a retroviral transfer plasmid of the invention.
  • a process for producing a retroviral packaging cell comprising the steps: introducing a retroviral transfer plasmid of the invention into a mammalian cell which expresses retroviral env and gag-pol genes, and optionally the rev gene.
  • the invention also provides the use of a retroviral packaging cell of the invention in the production of a retroviral vector.
  • the first, second, further and third (when present) nucleic acid molecules are expressed in the cell under conditions which are suitable for the production of lenti viral vectors comprising (ii), (iii) and (iv).
  • the cells will be cultured in a cell media, preferably in a liquid cell media. In some embodiments, the cells will be cultured in suspension.
  • Suitable conditions for performing the process of the invention are well known in the art (e.g. Benskey, M.J., Manfredsson, F.P. (2016). “Lentivirus Production and Purification”. In: Manfredsson, F. (eds) Gene Therapy for Neurological Disorders. Methods in Molecular Biology, vol 1382. Humana Press, New York, NY).
  • Step (B) relates to harvesting the retroviral vectors from the cell or from the cell media around the cell.
  • the retroviral vectors will be secreted into the cell media.
  • the cells could be removed from the cell media by, for example, centrifugation, filtration (e.g. tangential flow filtration), leaving the desired retroviral vectors in the cell supernatant.
  • the invention also provides a retroviral vector obtained or obtainable by a process of the invention.
  • the process/method steps are carried out (one after the other) in the order specified.
  • sequence comparison algorithm calculates the percentage sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Alignment of amino acid or nucleic acid sequences for comparison may be conducted, for example, by computer- implemented algorithms (e.g. GAP, BESTFIT, FASTA or TFASTA), or BLAST and BLAST 2.0 algorithms.
  • Percentage amino acid sequence identities and nucleotide sequence identities may be obtained using the BLAST methods of alignment (Altschul et al. (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402; and http://www.ncbi.nlm.nih.gov/BLAST). Preferably the standard or default alignment parameters are used.
  • blastp Standard protein-protein BLAST
  • blastp is designed to find local regions of similarity. When sequence similarity spans the whole sequence, blastp will also report a global alignment, which is the preferred result for protein identification purposes.
  • the standard or default alignment parameters are used.
  • the "low complexity filter” may be taken off.
  • Gapped BLAST in BLAST 2.0
  • PSI-BLAST in BLAST 2.0
  • the default parameters of the respective programs may be used.
  • MEGABLAST discontiguous- megablast, and blastn may be used to accomplish this goal.
  • the standard or default alignment parameters are used.
  • MEGABLAST is specifically designed to efficiently find long alignments between very similar sequences.
  • Discontiguous MEGABLAST may be used to find nucleotide sequences which are similar, but not identical, to the nucleic acids of the invention.
  • the BLAST nucleotide algorithm finds similar sequences by breaking the query into short subsequences called words. The program identifies the exact matches to the query words first (word hits). The BLAST program then extends these word hits in multiple steps to generate the final gapped alignments.
  • blastn is more sensitive than MEGABLAST. The most important reason that blastn is more sensitive than MEGABLAST is that it uses a shorter default word size (11). Because of this, blastn is better than MEGABLAST at finding alignments to related nucleotide sequences from other organisms.
  • the word size is adjustable in blastn and can be reduced from the default value to a minimum of 7 to increase search sensitivity.
  • discontiguous megablast uses an algorithm which is similar to that reported by Ma et al. (Bioinformatics. 2002 Mar; 18(3): 440-5). Rather than requiring exact word matches as seeds for alignment extension, discontiguous megablast uses non-contiguous word within a longer window of template.
  • the third base wobbling is taken into consideration by focusing on finding matches at the first and second codon positions while ignoring the mismatches in the third position.
  • the BLASTP 2.5.0+ algorithm may be used (such as that available from the NCBI) using the default parameters.
  • a BLAST Global Alignment program may be used (such as that available from the NCBI) using a Needleman-Wunsch alignment of two protein sequences with the gap costs: Existence 11 and Extension 1 .
  • sequence identity in the context of amino acid sequences may alternatively be replaced by “sequence similarity”.
  • similarity allows conservative substitutions of amino acid residues having similar physicochemical properties over a defined length of a given alignment. The percentage of similarity is determinable with any reasonable similarity-scoring matrix.
  • FIG. 1 Gene of interest expression during lentiviral vector production in the presence of shRNA plasmid EP2340.
  • Enhanced green fluorescent protein (EGFP) expression expressed as median fluorescence intensity multiplied by the percentage of EGFP- positive cells.
  • N three production replicates and two analytical replicates. Error bars indicate standard deviation.
  • FIG. 1 Gene of interest (GOI) expression per transducing unit in target cells transduced by lentiviral vectors produced in the presence of GOI-silencing shRNAs.
  • MFI median fluorescence intensity.
  • TU transducing units.
  • N three production replicates and two analytical replicates. Error bars indicate standard deviation.
  • Figure 4 Gene of interest expression during lentiviral vector production in the absence of shRNA plasmid EP2340.
  • Enhanced green fluorescent protein (EGFP) expression expressed as median fluorescence intensity multiplied by the percentage of EGFP- positive cells.
  • N three production replicates and two analytical replicates. Error bars indicate standard deviation.
  • FIG. 1 Gene of interest (GOI) expression per transducing unit in target cells transduced by lentiviral vectors produced in the absence of GOI-silencing shRNAs.
  • MFI median fluorescence intensity.
  • TU transducing units.
  • N three production replicates and two analytical replicates. Error bars indicate standard deviation.
  • Figure 7 Genetic constructs to simulate perfect gene of interest (GOI) knockdown and demonstrate the necessity of reversing the orientation of the GOI.
  • GOI perfect gene of interest
  • Figure 8 Example of a one-plasmid gene of interest-silencing system.
  • Figure 9 Example of a universal gene of interest silencing system.
  • a plasmid that encodes a lentiviral vector genome with the GOI in the opposite orientation to the viral RNA promoter; and a fragment of the PKR gene in the 3’ untranslated region (UTR) of the GOI; and an anti-PKR shRNA that targets both the PKR gene and the fragment of the PKR gene in the 3’ UTR of the GOI.
  • Figure 10 Effect of silencing toxic genes of interest during lentiviral vector (LVV) production.
  • A Anti-CD19 CAR expression during LVV production.
  • B Production cell viability during production of anti-CD19 CAR-encoding LVV.
  • C Production cell growth during production of anti-CD19 CAR-encoding LVV.
  • D Infectious titre of anti-CD19 CAR-encoding LVV.
  • E Production cell viability during production of BAX-encoding LVV.
  • F Production cell growth during production of BAX-encoding LVV.
  • G Physical titre of BAX-encoding LVV.
  • FIG 11. One-plasmid universal GOI-silencing system with various GOI promoters.
  • NTC non-transfected control.
  • FIG. 12 Generation of producer cell lines that encode anti-CD19 CAR within the GOI- silencing system.
  • A Density of cell cultures following transfection/integration of transfer plasmids. Average cell count calculated from two measurements, one from each of two replicate cell lines. Error bars indicate standard deviation.
  • B Viability of cell cultures following transfection/integration of transfer plasmids. Average cell viability calculated from two measurements, one from each of two replicate cell lines. Error bars indicate standard deviation.
  • C Infectious titre of lentiviral vectors (LVVs) prepared using producer cell lines. Infectious titration by ddPCR measurement of integrated vector copy number in transduced HEK293T cells.
  • LVV lentiviral vector
  • GOI lentiviral vector silencing idea
  • R2435 (SEQ ID NO: 12), the “current process”, which encodes an LVV genome with an enhanced green fluorescent protein (EGFP) as the GOI.
  • EGFP enhanced green fluorescent protein
  • R4751 SEQ ID NO: 13
  • Flip GOI(1) which encodes an LVV genome with an EGFP as the GOI.
  • the LVV genome and GOI were encoded in the opposite sense orientation to one another.
  • a rabbit alpha globin polyadenylation signal was encoded downstream of the LVV genome.
  • R4875 SEQ ID NO: 14
  • Flip GOI (2) which encodes an LVV genome with an EGFP as the GOI.
  • the LVV genome and GOI were encoded in the opposite sense orientation to one another.
  • a bovine growth hormone polyadenylation signal was encoded downstream of the LVV genome.
  • a plasmid was assembled to knock down EGFP and PKR (protein kinase R) genes.
  • This plasmid (EP2340 or “shRNA” plasmid, SEQ ID NO: 15) encodes two short hairpin RNAs (shRNAs): one targeted to the EGFP mRNA and the other targeted to the PKR mRNA.
  • the EGFP gene is encoded in the opposite sense direction to the LVV genome, it was expected that EGFP would be knocked down by the shRNA plasmid and the viral RNA would not be knocked down. If the GOI were not reversed, the EGFP gene and viral RNA would be encoded in the same sense direction as each other so it would be expected that both would be knocked down, hence the GOI was reversed in sense orientation.
  • the EGFP gene was encoded in the opposite sense direction to the LVV genome, the entire length of the GOI mRNA would have reverse complementarity to a section of the viral RNA. It was thus expected that a double-stranded RNA would be formed (comprising the GOI mRNA and the viral RNA), which would be expected to trigger a cell death cascade mediated by PKR. It is for this reason that an shRNA targeted to PKR was encoded within the shRNA plasmid (to inhibit the cell death cascade).
  • Table 1 Summary of the descriptions of the plasmids used. Plasmid sequences are disclosed in the Sequence Listing.
  • LVV production was performed by transfecting LVV transfer plasmid variants simultaneously with the shRNA plasmid (Table 1 and Table 2) into LVV packaging cells in the following combinations:
  • Table 2 Lentiviral vector production experimental procedure.
  • MFI median fluorescence intensity
  • Table 3 Flow cytometry experimental procedure. Finally, the MFI of the HEK 293T cells against which the LVV supernatants were titrated was measured to determine whether the shRNA affected GOI expression in the target cells and whether the modifications made to the LVV transfer plasmid affected GOI expression in the target cells (Table 3).
  • the first comparison between the current process and the gene-silenced process was between the levels of GOI expression during LVV production ( Figure 1).
  • SD 6.42 x10 5
  • infectious titres of LVV supernatants produced by the current process were compared to those of the gene-silenced process ( Figure 2).
  • GOI expression per transducing unit in model target cells was measured, comparing the current process to the flipped-GOI plasmids in the absence of the shRNA plasmid ( Figure 6).
  • shRNA plasmid alleviated this selective pressure as it would be expected to protect the production cells from double-stranded RNA either by the removal of one RNA strand by the anti-GOI shRNA (thus leaving single stranded RNA), or by the inhibition of the cellular response to the doublestranded RNA.
  • GOI gene products are known to be either directly toxic to mammalian cells or to interfere with lentiviral vector production:
  • BAX B-cell lymphoma 2-associated X-protein
  • the effects could include the following: Slow growth of production cells compared to the cells from which they were derived; An unacceptable level of production cell death during LVV production; Low LVV production compared to production cells that do not encode a toxic GOI; Reduced transduction efficiency of LVV that is produced e.g. coagulation factor VIII is known to impact the display of VSV-G (vesicular stomatitis virus G protein) on the surface of LVV particles, which reduces infectious titre without impacting physical titre (Radcliffe et al., Gene Ther., 2008 Feb; 15: 289-297). Silencing a toxic GOI during LVV production would thus be expected to ameliorate these issues.
  • VSV-G vesicular stomatitis virus G protein
  • an shRNA that targets the GOI is identified by a screen whereby production cells are transfected with the transfer plasmid and plasmids that encode various shRNA variants designed to target the toxic GOI and PKR.
  • GOI expression is measured by one or more of the following methods: measurement of GOI mRNA level by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR); measurement of GOI protein production by immunoblotting techniques; cell viability assay (if the GOI is toxic to the production cells, their death will imply GOI expression).
  • GOI expression is compared to a control that is transfected with the transfer plasmid and an shRNA plasmid that does not target the GOI and does target PKR.
  • the shRNA plasmid that is transfected into the cells that exhibited the lowest level of GOI expression compared to the control is selected as the shRNA plasmid for further use.
  • Production cells are transfected with the toxic GOI-encoding transfer plasmid and the top shRNA-encoding plasmid is identified in the above screen.
  • doxycycline is added to cell cultures to a final concentration of 1 pg/mL.
  • sodium butyrate is added to cell cultures to a final concentration of 5 mM.
  • LVV supernatants are harvested by removal of production cells by centrifugation as per Table 2.
  • Infectious titres of LVV supernatants are acquired by the measurement by qPCR (quantitative polymerase chain reaction) of the integrated vector copy number in transduced HEK293T cells, or by cell viability assay if the GOI is too toxic to permit titration by qPCR.
  • Physical titres of LVV supernatants are measured by RT-qPCR targeted to a packaged sequence, or by enzyme-linked immunosorbent assay (ELISA) targeted to p24.
  • the titre of the supernatant from cells transfected with the transfer plasmid and the GOI-targeted shRNA plasmid is compared to the titre of the supernatant from cells transfect with the transfer plasmid and a plasmid that encodes a plasmid that does not target the GOI.
  • An increase in viral vector titre is expected as a result of the inclusion of the GOI-targeted shRNA.
  • GOI expression in the transduced HEK293T cells is measured by one or more of the following methods: measurement of GOI mRNA level by RT-qPCR; measurement of GOI protein production by immunoblotting techniques; cell viability assay (if the GOI is toxic to the production cells, their death will imply GOI expression).
  • An increase in GOI expression in transduced HEK293T cells is expected as a result of the inclusion of the GOI-silencing shRNA during LVV production when compared to a control to which an shRNA that does not target the GOI was added.
  • CARs chimeric antigen receptors
  • GOI chimeric antigen receptors
  • they would also be expected to be displayed on the lentiviral vector surface, as it is an enveloped viral vector whose envelope is derived from the production cell membrane.
  • silencing these genes during lentiviral vector production would be beneficial as it would prevent the display of the GOI gene product on the lentiviral vector surface, making the end-product consistent, making downstream processing simpler and reducing the risk of immunogenicity.
  • a CAR gene (or gene of another membrane-displayed protein) is cloned into the LVV transfer plasmid of the GOI silencing system.
  • an shRNA that targets the GOI is identified by a screen whereby production cells are transfected with the transfer plasmid and plasmids that encode various shRNA variants designed to target the GOI and PKR.
  • GOI expression is measured by one or more of the following methods: measurement of GOI mRNA level by RT-qPCR; measurement of GOI protein production by immunoblotting techniques; cell viability assay (if the GOI is toxic to the production cells, their death will imply GOI expression).
  • GOI expression is compared to a control that is transfected with the transfer plasmid and an shRNA plasmid that does not target the GOI and does target PKR.
  • the shRNA plasmid that is transfected into the cells that exhibited the lowest level of GOI expression compared to the control is selected as the shRNA plasmid for further use.
  • viral vector is produced as follows: Production cells are transfected with the GOI- encoding transfer plasmid and the top shRNA-encoding plasmid identified in the above screen. Four to five hours after plasmid transfection, doxycycline is added to cell cultures to a final concentration of 1 pg/mL. Twenty-four hours later, sodium butyrate is added to cell cultures to a final concentration of 5 mM. Forty-eight hours later, LVV supernatants are harvested by removal of production cells by centrifugation as per Table 2.
  • GOI gene product display on viral vector surface is measured by immunoblotting techniques e.g. ELISA. It is expected that GOI gene product display on the lentiviral vector surface is reduced by the inclusion of a GOI-targeted shRNA during LVV production compared to a control to which an shRNA that does not target the GOI was added.
  • downstream processing of silencing a GOI that encodes a membrane-displayed protein is tested as follows: Downstream processing of LVV supernatants is performed according to a protocol that has been optimised for LVV that do not display the GOI gene product on the viral vector surface e.g. LVV that encodes EGFP. Efficiency of downstream processing is measured by comparison of LVV titre before and after downstream processing. The efficiencies of downstream processing of the following samples are compared: Membrane protein-encoding LVV produced in the presence of GOI-targeted shRNA; Membrane protein-encoding LVV produced in the absence of GOI-targeted shRNA; Non-membrane protein-encoding LVV.
  • Example 6 Testing the effect of silencing a toxic GOI during LW production
  • silencing a toxic GOI during LVV production would ameliorate the issues of: (1) low growth of production cells compared to the cells from which they were derived; (2) unacceptable level of production cell death during LVV production; and (3) low LVV production compared to production cells that do not encode a toxic GOI.
  • an anti-CD19 CAR gene and a B-cell lymphoma-associated X-protein (BAX) gene were inserted into gene-silencing LVV transfer plasmids ( Figure 9) and conventional LVV transfer plasmids (with the GOI in the same orientation as the LTRs; SEQ ID NOs: 24-27).
  • LVV production was performed with conventional and silenced anti-CD19 CAR and BAX plasmids, and the following were measured: anti-CD19 CAR expression during LVV production; production cell growth; production cell viability; infectious titre of anti-CD19 CAR LVV; and the physical titre of BAX LVV.
  • Anti-CD19 CAR expression was measured by staining live production cells with biotinylated recombinant protein L, then streptavidin-phycoerythrin, and then measurement of fluorescence levels by flow cytometry. This revealed that the silencing system reduced anti-CD19 CAR expression during LVV production by 55% (Figure 10A). Production cell growth and viability were measured by ViCell BLU Cell Viability Analyser (Beckman Coulter). This revealed that silencing anti-CD19 CAR during LVV production resulted in an increase in both production cell viability (Figure 10B) and growth (Figure 10C) during LVV production.
  • Example 7 Testing the “universal plasmid” LW GOI-silencing system with various transgene promoters
  • transfer plasmids were assembled as shown in Figure 9 with either SFFV, EFS or EF-1a promoter driving expression of the GOI (which was destabilised EGFP, i.e. ‘DEGFP’) (SEQ ID NOs: 18-23).
  • LVV production was performed by transfection of transfer plasmids into LVV packaging cells. DEGFP expression was monitored throughout production by flow cytometry. This revealed GOI knockdown in all cases ( Figure 11 A-C), confirming the function of the universal system with various promoters driving GOI expression.
  • LVV infectious titre was also measured by droplet digital PCR measurement of integrated vector copy number in transduced adherent HEK293T cells. This revealed that silencing DEGFP expression during LVV production resulted in a 42-56% decrease in LVV infectious titre (Figure 11 D).
  • Example 8 Testing the effect of integrating anti-CD19 CAR-encoding transfer plasmids into the genome of LW packaging cells
  • LVV producer cell lines that utilise the GOI-silencing system
  • conventional and silenced anti-CD19 CAR-encoding transfer plasmids were integrated into the genome of LVV packaging cells (LVPack13-14 cells) by transposase-mediated integration.
  • Integrated cells were cultured in growth media supplemented with antibiotic to which the transfected transfer plasmid encodes resistance, with twice-weekly monitoring of cell count and viability. This revealed that cells transfected/integrated with conventional antiCD 19 CAR-encoding transfer plasmid failed to recover; this is likely to be due to GOI toxicity ( Figure 12 A-B). These cells were thus discarded 17 days post-transfection. In contrast, cells transfected with silenced anti-CD19 CAR-encoding transfer plasmid recovered after transfection, indicating that the transfer plasmid can be integrated in the genome of the cell and that the GOI-silencing system can enable the recovery of otherwise non-recoverable producer cell lines ( Figure 12 A-B).
  • LVVs were produced by addition of doxycycline to producer cell line cultures.
  • Example 9 Testing the performance of the GOI-silencing system in four-plasmid- transfection-based LW production
  • the “universal plasmid” ( Figure 9) GOI-silencing system was tested in a four-plasmid transfection production modality in suspension HEK293 cells (WXATUS0028 cell line).
  • Cells were transfected with plasmids encoding VSV-G, gag-pol, and Rev, and one of several transfer plasmids, either conventional (with the GOI in the same orientation as the LTRs) or GOI-silencing ( Figure 9; SEQ ID NOs: 19, 22, 24 or 25).
  • Either DEGFP or anti-CD19 CAR were used as the GOI.
  • Cell growth and viability were measured at the point of LVV harvest.
  • DEGFP expression level was measured throughout production at 24-hour intervals.
  • Anti-CD19 CAR expression level was measured at the point of LVV harvest.

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Abstract

L'invention concerne des procédés de production de vecteurs rétroviraux comprenant un transgène. Le procédé comprend l'expression dans une cellule : (i) d'un plasmide vecteur rétroviral comportant un transgène, le transgène étant en orientation inverse dans le plasmide ; (ii) d'un inhibiteur de l'ARN du transgène ; et (iii) d'un inhibiteur de la réponse à la mort cellulaire. L'invention concerne également des plasmides de transfert rétroviraux, des kits et des lignées cellulaires de production destinés à être utilisés dans les procédés de l'invention.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018209216A1 (fr) * 2017-05-12 2018-11-15 University Of Massachusetts Système de production de vecteurs viraux
WO2022225973A1 (fr) * 2021-04-20 2022-10-27 The Regents Of The University Of California Cellules d'encapsidation présentant des inactivations génétiques ciblées améliorant les titres des vecteurs rétroviraux
WO2023062366A1 (fr) * 2021-10-12 2023-04-20 Oxford Biomedica (Uk) Limited Vecteurs rétroviraux
WO2023062363A1 (fr) * 2021-10-12 2023-04-20 Oxford Biomedica (Uk) Limited Vecteurs lentiviraux

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4157326A (en) 1977-11-28 1979-06-05 Monsanto Company Preparation of glass concentrate capsules for reinforcement of thermoplastics

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018209216A1 (fr) * 2017-05-12 2018-11-15 University Of Massachusetts Système de production de vecteurs viraux
WO2022225973A1 (fr) * 2021-04-20 2022-10-27 The Regents Of The University Of California Cellules d'encapsidation présentant des inactivations génétiques ciblées améliorant les titres des vecteurs rétroviraux
WO2023062366A1 (fr) * 2021-10-12 2023-04-20 Oxford Biomedica (Uk) Limited Vecteurs rétroviraux
WO2023062363A1 (fr) * 2021-10-12 2023-04-20 Oxford Biomedica (Uk) Limited Vecteurs lentiviraux

Non-Patent Citations (14)

* Cited by examiner, † Cited by third party
Title
ALTSCHUL ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389
ALTSCHUL ET AL.: "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402, XP002905950, Retrieved from the Internet <URL:http://www.ncbi.nlm.nih.gov/BLAST> DOI: 10.1093/nar/25.17.3389
BENSKEY, M.J.MANFREDSSON, F.P.: "Gene Therapy for Neurological Disorders. Methods in Molecular Biology", vol. 1382, 2016, HUMANA PRESS, article "Lentivirus Production and Purification"
BLANCO ET AL., THE JOURNAL OF BIOCHEMISTRY, vol. 278, no. 2, 2003, pages 1086 - 1093
CUSACK ET AL., BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 79, 1 January 2023 (2023-01-01), pages 129047
FINKELSTEIN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 110, no. 18, 2013, pages 7306 - 7311
HU PEIRONG ET AL: "Superior lentiviral vectors designed for BSL-0 environment abolish vector mobilization", GENE THERAPY, NATURE PUBLISHING GROUP, LONDON, GB, vol. 25, no. 7, 6 September 2018 (2018-09-06), pages 454 - 472, XP036847146, ISSN: 0969-7128, [retrieved on 20180906], DOI: 10.1038/S41434-018-0039-2 *
JAMMI, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 308, no. 608512-97-6, 15 August 2003 (2003-08-15), pages 50 - 57
KAADT ET AL., MOLECULAR THERAPY NUCLEIC ACIDS, vol. 14, November 2018 (2018-11-01), pages 318 - 328
MA ET AL., BIOINFORMATICS, vol. 18, no. 3, March 2002 (2002-03-01), pages 440 - 5
PERNOD GILLES ET AL: "Increasing lentiviral vector titer using inhibitors of protein kinase R", vol. 36, no. 4, 1 January 2004 (2004-01-01), XP093008586, Retrieved from the Internet <URL:https://www.future-science.com/doi/pdf/10.2144/04364BM02> *
RADCLIFFE ET AL., GENE THER., vol. 15, February 2008 (2008-02-01), pages 289 - 297
SEVIER ET AL., MOL. BIOL. CELL, vol. 11, no. 1, January 2000 (2000-01-01), pages 13 - 22
SHUNIT GAL-BEN-ARI ET AL: "PKR: A Kinase to Remember", FRONTIERS IN MOLECULAR NEUROSCIENCE, vol. 11, 9 January 2019 (2019-01-09), pages 1 - 20, XP055731345, DOI: 10.3389/fnmol.2018.00480 *

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