Enhanced lentiviral vector production

Abstract

A modified U1 snRNA that has been modified to bind to a nucleotide sequence within the packaging region of a lentiviral vector genome sequence.

Description

[Technical Field]

[0001] The present invention relates to the production of lentiviral vectors in eukaryotic cells. More particularly, the present invention relates to the design of modified U1 snRNA and the co-expression of modified U1 snRNA during the production of lentiviral vectors to increase output titers. [Background technology]

[0002] The development and production of viral vectors for vaccines and human gene therapy over the past several decades has been well documented in the scientific literature and patents. The use of engineered viruses to deliver transgenes for therapeutic effect is widespread. Modern gene therapy vectors based on RNA viruses such as gamma-retroviruses and lentiviruses (Muhlebach, MD, et al., 2010, Retroviruses: Molecular Biology, Genomics and Pathogenesis, 13:347-370; Antoniou, MN, Skipper, KA & Anakok, O., 2013, Hum. Gene Ther., 24:363-374), and DNA viruses such as adenoviruses (Capasso, C. et al., 2014, Viruses, 6:832-855) and adeno-associated viruses (AAV) (Kotterman, MA & Schaffer, DV, 2014, Nat. Rev. Genet., 15:445-451), are increasingly showing promise in a number of human disease indications. These include ex vivo modification of patient cells for hematological pathologies (Morgan, RA & Kakarla, S., 2014, Cancer J., 20:145-150; Touzot, F. et al., 2014, Expert Opin. Biol. Ther., 14:789-798), as well as ophthalmological diseases (Balaggan, KS & Ali, RR, 2012, Gene Ther., 19:145-153), cardiovascular diseases (Katz, MG et al., 2013, Hum. Gene Ther., 24:914-927), neurodegenerative diseases (Coune, PG, Schneider, BL & Aebischer, P., 2012, Cold Spring Harb. Perspect. Med., 4:a009431) and tumor therapy (Pazarentzos, E. & Mazarakis, ND, 2014, Adv. Exp. Med Biol., 818:255-280).As the success of these approaches in clinical trials begins to build toward regulatory approval and commercialization, attention is being drawn to new obstacles in the large-scale production of Good Manufacturing Practice (GMP)-grade vector material (Van der Loo JCM, Wright JF., 2016, Human Molecular Genetics, 25(R1):R42-R52).

[0003] A means of overcoming this challenge is to find new methods for maximizing titers during viral vector production. Thus, there is a need in the art for alternative methods of producing viral vectors that help address the known challenges associated with large-scale production of GMP-grade vector material.

[0004] Common methods for viral vector production involve transfecting primary cells or mammalian / insect cell lines with vector DNA components, followed by recovery of crude vector from the medium and / or cells after a limited incubation period (Merten, OW., Schweizer, M., Chahal, P., & Kamen, AA, 2014, Pharmaceutical Bioprocessing, 2:183-203). In other cases, producer cell lines (PrCLs; in which all necessary vector component expression cassettes are stably integrated into the producer cell DNA) are used in transfection-independent approaches, which are advantageous for large-scale production. The efficiency of lentiviral vector production is typically affected by several factors in the "upstream" stages, including: [1] the viral serotype / pseudotype used, [2] the composition and size of the transgenic sequence, [3] the media composition / gassing / pH, [4] the transfection reagent / process, [5] the timing of chemical induction and vector harvest, [6] the fragility / viability of the cells, [7] the shear forces of the bioreactor, and [8] impurities. Clearly, there are other factors to consider in the "downstream" purification / concentration stages (Merten, OW. et al., 2014, Pharmaceutical Bioprocessing, 2:237-251).

[0005] One important aspect of optimization is the relative abundance of lentiviral vector components [GagPol, envelope, rev, and vector genomic RNA (vRNA)] during upstream production. For approaches requiring transient transfection of plasmid DNA encoding these components, the optimal "mass" ratio of these plasmids is typically identified during optimization. The optimal ratio can also be effectively obtained by screening a large number of PrCLs containing all of the components stably integrated into the host DNA. PrCLs exhibiting the highest yields are clones containing expression cassettes at loci that confer expression of each individual component at or near the optimal ratio. We note that the genomic vRNA component can often be a limiting factor in PrCLs. This is supported by reports that high-titer production by PrCL clones correlates with high copy numbers of the vector genomic cassette (Sheridan, PL et al., 2000, Mol. Ther., 2(3):262-275). Typically, the maximum proportion of plasmid DNA is approached at the optimal ratio of component plasmids during transient transfection, even when considering plasmid size. This indicates that the production of genomic vRNA during lentiviral vector production is a key factor in implementing an efficient lentiviral vector production approach.

[0006] Splicing and polyadenylation are essential processes for mRNA maturation, particularly in higher eukaryotes, where most protein-coding transcripts contain multiple introns. Elements within mRNA required for splicing include a 5' splice donor signal, a sequence surrounding the branch point, and a 3' splice acceptor signal. These three elements interact with the spliceosome, which is composed of five small nuclear RNAs (snRNAs), including the U1 snRNA, and associated nuclear proteins (snRNPs). U1 snRNA is expressed by a polymerase II promoter and is present in most eukaryotic cells (Lund et al., 1984, J. Biol. Chem., 259:2013-2021). The human U1 snRNA (small nuclear RNA) is 164 nt long and has a distinct four-stem-loop structure (West, S., 2012, Biochemical Society Transactions, 40:846-849). U1 snRNA contains a short sequence at its 5' end that is broadly complementary to the 5' splice donor site at exon-intron junctions. By base pairing with the 5' splice donor site, U1 snRNA is involved in splice site selection and spliceosome assembly. One of the known functions of U1 snRNA, other than splicing, is the regulation of 3' end mRNA processing. It suppresses premature polyadenylation (polyA) at early polyA signals (especially within introns).

[0007] The suppression of poly(A) sites within intronic sequences in cellular genes by recruitment of endogenous U1 snRNA has been well characterized (Kaida, D. et al., 2010, Nature, 468:664-8). Other researchers have also used modified U1 snRNA as a research tool to analyze the mechanism of suppression of premature polyadenylation in the 5' LTR during HIV-1 replication (Ashe, MP, Pearson, LH, & Proudfoot NJ, 1997, EMBO J., 16:5752-63; Ashe, MP, Furger, A. & Proudfoot, NJ, 2000, RNA, 6:170-7; Furger, A., Monks, J. & Proudfoot, NJ, 2001, J. Virol., 75:11735-46).

[0008] HIV-1 encodes identical poly(A) signals within the R regions of both the 5'LTR and 3'LTR, and the 3'LTR poly(A) is active and utilized to terminate all pre-mRNA transcription events. To avoid premature termination at the poly(A) site in the 5'LTR (i.e., termination immediately after transcription initiation), endogenous U1 snRNA binding to the major splice donor (approximately 200 bases downstream) suppresses the activity of this poly(A) site, allowing transcription to continue to the end of the proviral cassette. Mutation of the major splice donor activates the poly(A) site in the 5'LTR, but it has been shown that this repression can be restored when modified U1 snRNA (targeting sequences adjacent to the major splice donor) is coexpressed. However, poly(A) site suppression depends on the proximity of the U1 snRNA to the poly(A) site, and much of the work performed has been done in artificial "minigene" expression cassettes that lack many of the other RNA sequences found in wild-type HIV-1 or in HIV-1-based lentiviral vectors.

[0009] The manipulation and use of U1 snRNA, known in the art as "U1 interference" or "U1i," has been used to develop approaches to suppress gene expression (Beckley, SA et al., 2001, Mol. Cell Biol., 21:2815-25; Fortes, P. et al., 2003, Proc. Nat. Acad. Sci. USA, 100:8264-9). The mechanism by which U1i functions is by inhibiting the correct placement and processing of pre-mRNA polyadenylation, resulting in the production of unstable mRNA and reduced protein levels. Inhibition of polyadenylation requires the localization of modified U1 snRNP particles to the 3'-terminal exon of the target transcript. The nucleic acid sequence of the U1 snRNA gene is modified so that U1 snRNA binds to a selected sequence rather than the 5' splice donor site sequence used by U1 snRNA to initiate splicing of target genes. Inhibition of polyadenylation is achieved by base-pairing the 5' end of the modified U1 snRNA in the assembled RNP complex with a selected complementary region in the target pre-mRNA, and has been used as an antiviral approach (Blaquez, L. & Fortes, P., 2015, Adv. Exp. Med. Biol., 848:51-69), including against HIV-1 (Sajic, R. et al., 2007, Nucleic Acids Res., 35:247-55; Knoepfel, SA et al., 2012, Antiviral Res., 94:208-16).

[0010] Other researchers have shown that binding of endogenous or modified U1 snRNA to the HIV-1 consensus or non-consensus 5' splice site can alter viral RNA stability (Lutzelberger, M. et al., 2006, J. Biol. Chem., 281:18644-51). However, the report suggests that this effect is specific to a short novel intron found within the HIV-1 pol region; such a sequence is completely absent from vector genome sequences based on HIV-1 lentiviral vectors.

[0011] The manipulation and use of U1 snRNA to increase lentiviral vector titer during lentiviral vector production was not known at the filing date of this application. Summary of the Invention

[0012] Overview of the invention The present inventors have surprisingly found that the output titer of lentiviral vectors can be increased by co-expressing a non-coding RNA based on U1 snRNA that has been modified so that it no longer targets the endogenous sequence (splice donor site) but now targets a sequence within the vRNA molecule. The present invention relates to such modified U1 snRNA and a novel method for increasing the production titer of lentiviral vectors. This approach consists of co-expressing the modified U1 snRNA with other vector components during vector production. The modified U1 snRNA is designed such that binding to the consensus splice donor site is eliminated by replacing the consensus splice donor site with a heterologous sequence complementary to the target sequence within the vector genome vRNA. The present invention describes optimal features and various modes of application of the modified U1 snRNA, including the length, design, and expression modes of the target sequence and complementarity.

[0013] In one aspect, the present invention provides a modified U1 snRNA that has been modified to bind to a nucleotide sequence within the packaging region of a lentiviral vector genome sequence.

[0014] In one aspect, the present invention provides a modified U1 snRNA that has been modified to be complementary to a nucleotide sequence within the packaging region of a lentiviral vector genome sequence.

[0015] In some embodiments, the modified U1 snRNA is modified to introduce a heterologous sequence that is complementary to the nucleotide sequence.

[0016] In some embodiments, the modified U1 snRNA is modified at the 5' end to introduce the heterologous sequence within 9 nucleotides from positions 3-11.

[0017] In some embodiments, the modified U1 snRNA is modified at the 5' end to introduce the heterologous sequence within the natural splice donor annealing sequence.

[0018] In some embodiments, 1 to 9 nucleotides of the native splice donor annealing sequence are replaced by the heterologous sequence, hi one aspect, nucleotides 1 to 11 comprising the native splice donor annealing sequence are replaced by a heterologous sequence complementary to the nucleotide sequence.

[0019] In some embodiments, the modified U1 snRNA is modified at the 5' end to replace the sequence comprising the natural splice donor annealing sequence with a heterologous sequence complementary to the nucleotide sequence.

[0020] In some embodiments, the heterologous sequence comprises at least 9 nucleotides complementary to the nucleotide sequence.

[0021] In some embodiments, the heterologous sequence comprises 15 nucleotides complementary to the nucleotide sequence.

[0022] In some embodiments, the packaging region of the lentiviral vector genome sequence is from the beginning of the 5' U5 domain to the end of the gag gene-derived sequence.

[0023] In some embodiments, the nucleotide sequence is located within the 5'U5 domain, the PBS element, the SL1 element, the SL2 element, the SL3ψ element, the SL4 element, and / or a sequence derived from the gag gene.

[0024] In some embodiments, the nucleotide sequence is located within the SL1, SL2 and / or SL3ψ element.

[0025] In some embodiments, the nucleotide sequence is located within the SL1 and / or SL2 element.

[0026] In some embodiments, the nucleotide sequence is located within the SL1 element.

[0027] In some embodiments, the modified U1 snRNA is a modified U1A snRNA or a modified U1A snRNA variant.

[0028] In some embodiments, the first two nucleotides at the 5' end of the modified U1 snRNA are not AU.

[0029] In some embodiments, the lentiviral vector is derived from an HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or visna lentivirus.

[0030] In some embodiments, the lentiviral vector is derived from HIV-1, HIV-2, or EIAV.

[0031] In some embodiments, the lentiviral vector is derived from HIV-1.

[0032] In some embodiments, the lentiviral vector is derived from SIV.

[0033] In another aspect, the present invention provides an expression cassette comprising a nucleotide sequence encoding a modified U1 snRNA of the present invention.

[0034] In another aspect, the present invention provides a cell for producing a lentiviral vector comprising nucleotide sequences encoding vector components including gag, env, rev and the RNA genome of a lentiviral vector, and at least one nucleotide sequence encoding a modified U1 snRNA of the present invention.

[0035] In another aspect, the present invention provides a cell comprising a modified U1 snRNA according to the present invention.

[0036] In some embodiments, the cell further comprises a nucleotide sequence encoding the RNA genome of a lentiviral vector.

[0037] In some embodiments, the cells further comprise a nucleotide sequence encoding a nucleotide of interest.

[0038] In some embodiments, the nucleotide of interest provides a therapeutic effect.

[0039] In some embodiments, the nucleotide of interest encodes an enzyme, a cofactor, a cytokine, a chemokine, a hormone, an antibody, an antioxidant molecule, an engineered immunoglobulin-like molecule, a single-chain antibody, a fusion protein, an immune co-stimulatory molecule, an immunomodulatory molecule, a chimeric antigen receptor, a trans-domain negative mutant of a target protein, a toxin, a conditional toxin, an antigen, a transcription factor, a structural protein, a reporter protein, a subcellular localization signal, a tumor suppressor protein, a growth factor, a membrane protein, a receptor, a vasoactive protein or peptide, an antiviral protein or ribozyme, or derivatives thereof, or a microRNA.

[0040] In some embodiments, the nucleotide of interest encodes a molecule useful for treating a disorder selected from the following: (i) Disorders responsive to the following: cytokine and cell proliferation / differentiation activity; immunosuppressant or immunostimulatory activity (e.g., for the treatment of immune deficiencies, including infection with the human immunodeficiency virus, for the regulation of lymphocyte proliferation, for the treatment of cancer (hereinafter referred to as cancer) and many autoimmune diseases, and for the prevention of transplant rejection or the induction of tumor immunity); regulation of hematopoiesis (e.g., for the treatment of bone marrow or lymphatic system diseases); promotion of growth of bone, cartilage, tendon, ligament, and nerve tissue (e.g., for the treatment of wound healing, burns, ulcers, and periodontal disease and neurodegeneration); inhibition or activation of follicle-stimulating hormone (regulation of fertility); chemotaxis / chemokine etic activity (e.g., for recruiting specific cell types to sites of injury or infection); hemostatic and thrombolytic activity (e.g., for the treatment of hemophilia and stroke); anti-inflammatory activity (e.g., for the treatment of septic shock or Crohn's disease); macrophage-inhibitory and / or T-cell-inhibitory activity, and therefore anti-inflammatory activity; anti-immune activity (i.e., inhibitory effects on cellular and / or humoral immune responses, including responses not related to inflammation); inhibition of the ability of macrophages and T cells to adhere to extracellular matrix components and fibronectin, and upregulated Fas receptor expression in T cells; (ii) malignant disorders, such as cancer, leukemia, benign and malignant tumor growth, invasion and spread, angiogenesis, metastasis, ascites and malignant pleural effusion; (iii) autoimmune diseases, such as arthritis, e.g., rheumatoid arthritis, hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other diseases; (iv) vascular diseases, such as arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome, cardiovascular effects, peripheral vascular disease, migraine and aspirin-dependent antithrombosis, stroke, cerebral ischemia, ischemic heart disease or other diseases; (v) diseases of the gastrointestinal tract, such as peptic ulcer, ulcerative colitis, Crohn's disease and other diseases; (vi) liver disease, e.g., liver fibrosis, cirrhosis; (vii) inherited metabolic disorders, such as phenylketonuria (PKU), Wilson's disease, organic acidemias, urea cycle disorders, cholestasis, and other diseases; (viii) kidney and urinary diseases, for example, thyroiditis or other glandular diseases, glomerulonephritis or other diseases; (ix) ear, nose, and throat disorders, such as otitis or other ENT diseases, dermatitis or other skin diseases; (x) dental and oral disorders, e.g., periodontal disease, periodontitis, gingivitis or other dental / oral diseases; (xi) testicular disease, e.g., orchitis or epididymo-orchitis, infertility, testicular trauma or other testicular disease; (xii) gynecological diseases, such as placental insufficiency, placental failure, habitual miscarriage, eclampsia, preeclampsia, endometriosis and other gynecological diseases; (xiii) ophthalmological disorders, for example, Leber congenital amaurosis (LCA), e.g., LCA10, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, glaucoma, for example, open-angle glaucoma and juvenile congenital glaucoma, intraocular inflammation, for example, retinitis or cystoid macular edema, sympathetic ophthalmia, scleritis, retinitis pigmentosa, macular degeneration, for example, age-related macular degeneration (AMD) and juvenile macular degeneration, for example, Best disease, Best vitelliform macular degeneration, Stargardt disease, Usher syndrome, Doyne honeycomb retinal dystrophy, Sorby yellodendronate macular dystrophy, juvenile retinoschisis, cone-rod dystrophy, corneal dystrophy, Fuchs' dystrophy, Leber congenital amaurosis, Leber hereditary optic neuropathy (LHON), Adie's syndrome, Oguchi's disease, degenerative fundus diseases, ocular trauma, ocular inflammation caused by infection, proliferative vitreoretinopathy, acute ischemic optic neuropathy, excessive scarring, such as after glaucoma filtration surgery, reactions to ocular implants, corneal transplant graft rejection, and other ophthalmic diseases, such as diabetic macular edema, retinal vein occlusion, RLBP1-related retinal dystrophy, choroideremia, and color vision disorders; (xiv) neurological disorders and neurodegenerative disorders, for example, Parkinson's disease, complications and / or side effects of treatment for Parkinson's disease, AIDS-related dementia syndrome, HIV-associated encephalopathy, Devic's disease, Sydenham's chorea, Alzheimer's disease and other degenerative diseases, CNS pathologies or disorders, stroke, post-polio syndrome, psychiatric disorders, myelitis, encephalitis, subacute sclerosing panencephalitis, encephalomyelitis, acute neurological disorders, subacute neurological disorders, chronic neurological disorders, Fabry's disease, Gaucher's disease, cystinosis, Pompe's disease, heterochromatin disorders, leukodystrophy, Wiskott-Aldrich syndrome, adrenoleukodystrophy, beta thalassemia, sickle cell disease, Guillain-Barré syndrome, Sydenham chorea, myasthenia gravis, pseudotumor cerebri, Down syndrome, Huntington's disease, CNS compression or CNS trauma or infection of the CNS, muscle atrophy and muscular dystrophies, diseases, conditions or disorders of the central and peripheral nervous system, motor neuron diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy, spinal cord and avulsion injuries; and (xv) For suppressing or inhibiting humoral and / or cellular immune responses to prevent and / or treat graft rejection in the case of cystic fibrosis, mucopolysaccharidoses such as Sanfilippo syndrome A, Sanfilippo syndrome B, Sanfilippo syndrome C, Sanfilippo syndrome D, Hunter syndrome, Hurler-Scheie syndrome, Morquio syndrome, ADA-SCID, X-linked SCID, X-linked chronic granulomatous disease, porphyria, hemophilia A, hemophilia B, post-traumatic inflammation, bleeding, coagulation and acute phase response, cachexia, anorexia, acute infection, septic shock, infection, diabetes, complications or side effects of surgery, complications and / or side effects of bone marrow transplantation or other transplants, complications and side effects of gene therapy, e.g., due to infection with a viral carrier or AIDS, transplantation of natural or artificial cells, tissues and organs, e.g., cornea, bone marrow, organ, lens, pacemaker, natural or artificial skin tissue.

[0041] In another aspect, the present invention provides stable or transient production cells for producing lentiviral vectors comprising at least one nucleotide sequence encoding a modified U1 snRNA of the present invention.

[0042] In another embodiment, a. introducing into a cell nucleotide sequences encoding vector components including gag, env, rev and the RNA genome of a lentiviral vector, and at least one nucleotide sequence encoding a modified U1 snRNA of the present invention; b. optionally, selecting cells containing the vector components and the nucleotide sequence encoding at least one modified U1 snRNA; c. culturing the cells under conditions in which the vector components are co-expressed with the modified U1 snRNA and a lentiviral vector is produced. The present invention provides a method for producing a lentiviral vector as described herein, comprising:

[0043] In another aspect, the present invention provides a lentiviral vector produced by the production method of the present invention.

[0044] In another aspect, the present invention provides the use of a modified U1 snRNA of the invention or an expression cassette of the invention for the production of a lentiviral vector.

[0045] In another aspect, the present invention provides a lentiviral vector produced in the presence of a modified U1 snRNA of the present invention, wherein the lentiviral vector comprises an inactivated major splice donor site in the RNA genome of the lentiviral vector.

[0046] In some embodiments, the lentiviral vector is derived from an HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or visna lentivirus.

[0047] In some embodiments, the lentiviral vector is derived from HIV-1, HIV-2, or EIAV.

[0048] In some embodiments, the lentiviral vector is derived from HIV-1.

[0049] In some embodiments, the lentiviral vector is derived from SIV.

[0050] In some embodiments described herein, the major splice donor site in the RNA genome of the lentiviral vector is inactivated.

[0051] In some embodiments, the major splice donor site and the cryptic splice donor site 3' to the major splice donor site in the RNA genome of the lentiviral vector are inactivated.

[0052] In some embodiments, the lentiviral vector is a third generation lentiviral vector.

[0053] In some embodiments, the major splice donor site in the RNA genome of the lentiviral vector is inactivated, a cryptic splice donor site 3' to the major splice donor site is inactivated, and the nucleotide sequence is for use in a tat-independent lentiviral vector.

[0054] In some embodiments, the major splice donor site in the RNA genome of the lentiviral vector is inactivated, a cryptic splice donor site 3' to the major splice donor site is inactivated, and the nucleotide sequence is produced in the absence of tat.

[0055] In some embodiments, the major splice donor site in the RNA genome of the lentiviral vector is inactivated, a cryptic splice donor site 3' to the major splice donor site is inactivated, and the nucleotide sequence is transcribed independently of tat.

[0056] In some embodiments, the major splice donor site in the RNA genome of the lentiviral vector is inactivated, and a cryptic splice donor site 3' to the major splice donor site is inactivated, and the nucleotide sequence is for use in a U3-independent lentiviral vector.

[0057] In some embodiments, the major splice donor site in the RNA genome of the lentiviral vector is inactivated, a cryptic splice donor site 3' to the major splice donor site is inactivated, and the nucleotide sequence is transcribed independently of the U3 promoter.

[0058] In some embodiments, the major splice donor site in the RNA genome of the lentiviral vector is inactivated, a cryptic splice donor site 3' to the major splice donor site is inactivated, and the nucleotide sequence is transcribed by a heterologous promoter.

[0059] In some embodiments, the cryptic splice donor site is the first cryptic splice donor site 3' to the major splice donor site.

[0060] In some embodiments, the cryptic splice donor site is within 6 nucleotides of the major splice donor site.

[0061] In some embodiments, the major splice donor site and the cryptic splice donor sites are mutated or deleted.

[0062] In one embodiment, the present invention provides a nucleotide sequence encoding the RNA genome of a lentiviral vector, wherein the nucleotide sequence prior to inactivation of splice sites comprises the sequence set forth in any of SEQ ID NOs: 1, 3, 4, 9, 10 and / or 13. The nucleotide sequence may comprise a sequence containing a mutation or deletion compared to the sequence set forth in any of SEQ ID NOs: 1, 3, 4, 9, 10 and / or 13. In one embodiment, the sequence comprises SEQ ID NO: 13.

[0063] In one embodiment, the nucleotide sequence comprises an inactivated major splice donor site which, when not inactivated, has a cleavage site immediately upstream of nucleotide 1 (SEQ ID NO: 13) of the major splice donor region.

[0064] In one embodiment, the nucleotide sequence comprises an inactivated major splice donor site and an inactivated cryptic splice donor site which, when not inactivated, has a cleavage site immediately upstream of nucleotide 1 and between nucleotides 4 and 5, which correspond to the nucleotides of the major splice donor region (SEQ ID NO: 13).

[0065] In some embodiments, the nucleotide sequence encoding the RNA genome of the lentiviral vector comprises an inactivated major splice donor site which, when not inactivated, has a cleavage site between nucleotides corresponding to nucleotides 13 and 14 of SEQ ID NO:1.

[0066] In some embodiments, the nucleotide sequence of the major splice donor site before inactivation comprises the sequence set forth in SEQ ID NO:4.

[0067] In some embodiments, the nucleotide sequence of the cryptic splice donor site before inactivation comprises the sequence set forth in SEQ ID NO:10.

[0068] In some embodiments, the nucleotide sequence encoding the RNA genome of the lentiviral vector comprises an inactivated cryptic splice donor site which, when not inactivated, has a cleavage site between nucleotides corresponding to nucleotides 17 and 18 of SEQ ID NO:1.

[0069] In some embodiments, the nucleotide sequence encoding the RNA genome of the lentiviral vector comprises the sequence set forth in any of SEQ ID NOs: 2, 5, 6, 7, 8, 11, 12 and / or 14.

[0070] In a preferred embodiment, the nucleotide sequence comprises the sequence set forth in SEQ ID NO:14.

[0071] In some embodiments, the nucleotide sequence encoding the RNA genome of the lentiviral vector does not include the sequence set forth in SEQ ID NO:9.

[0072] In some embodiments, splicing activity from major and cryptic splice donor sites in the RNA genome of the lentiviral vector is inhibited or eliminated.

[0073] In some embodiments, splicing activity from major and cryptic splice donor sites in the RNA genome of the lentiviral vector is suppressed or eliminated in transfected or transduced cells.

[0074] In some embodiments, the nucleotide sequence encoding the RNA genome of the lentiviral vector is operably linked to a nucleotide sequence encoding a modified U1 snRNA.

[0075] In one embodiment, the nucleotide sequence encoding the modified U1 snRNA is present on a different nucleotide sequence, eg, a different plasmid, than the nucleotide sequence encoding the RNA genome of the lentiviral vector.

[0076] In one embodiment, the nucleotide sequence according to the invention is for use in a tat-independent lentiviral vector system. In one embodiment, the lentiviral vector system may be a third generation lentiviral vector system.

[0077] In one embodiment, the nucleotide sequence may be suitable for use in a lentiviral vector in a tat-independent system for vector production. As described herein, third-generation lentiviral vectors are tat-independent, and the nucleotide sequence according to the present invention may be used in the context of a third-generation lentiviral vector. For clarity, the term "tat-independent" is understood to mean that the HIV-1 U3 promoter used to drive transcription of the vector genome cassette is replaced by a heterologous promoter. In one embodiment of the present invention, tat is not provided in the lentiviral vector production system. For example, tat is not provided in trans. In one embodiment, the cells or vectors or vector production systems described herein do not contain tat protein. [Brief explanation of the drawings]

[0078] [Figure 1]Schematic diagram of the U1 snRNA molecule and an example of how the targeting sequence used in the present invention can be modified. U1 snRNA, an endogenous noncoding RNA, binds to a consensus splice donor site (5'-MAGGURR-3') via the natural splice donor targeting sequence 5'-(AC)UUACCUG-3' (highlighted in gray) during the early stages of intron splicing. Stem-loop I binds to the U1A-70K protein, which has been shown to be important for poly(A) repression. Stem-loop II binds to the U1A protein, and the 5'-AUUUGUGG-3' sequence binds to the Sm protein, which, together with stem-loop IV, is important for U1 snRNA processing. In the present invention, the modified U1 snRNA is modified at the site of the natural splice donor targeting sequence to introduce a heterologous sequence complementary to a target sequence within a vector genome vRNA molecule. In this figure, the example shown targets the modified U1 snRNA to 15 nucleotides (256-270 based on the first nucleotide of the vector genome molecule 256U1) of the standard HIV-1 lentiviral vector genome (located within the SL1 loop in the case of the packaging signal). [Figure 2] The increased lentiviral vector titer conferred by modified U1 snRNA is independent of poly(A) site suppression in the 5' LTR of the vector genome. [A] To evaluate the effects of two poly(A) signal mutants (pAM1 = AAUAAA>AACAAA; pAKO = deletion of AAUAAA) and the wild-type poly(A) signal (wt pA = AAUAAA) on transcriptional readthrough of the HIV-1 poly(A) site, a GFP-poly(A) luciferase reporter cassette was designed. Readthrough of the HIV-1 poly(A) signal was measured by luciferase activity normalized to GFP expression. [B] Vector particles were generated using a standard lentiviral vector genome (STD-LV) and two lentiviral vector genomes containing different 5' LTR poly(A) signal mutants (Δ5'pA-LVpAM1 or pAM2) in the absence (NegCtrl) or presence (NegCtrl) of modified U1 snRNA (256_U1; supplied in parallel during production), and then titered. [Figure 3] The increased lentiviral vector titer conferred by modified U1 snRNA does not require a functional U1A-70K or U1A protein binding loop. [A] U1 snRNA was modified to target the 256-270 region of the LV genome or two sites in the lacZ sequence (negative control). Modified U1A snRNAs were engineered to contain mutations known to disrupt U1A-70K protein, U1A protein, or sm protein binding (sequences shown). Modified U1A5, U1A6, and U1A7 snRNA mutants were also engineered. [B] Effect of various modified U1 snRNAs supplied in trans during production on LV-GFP titer. [Figure 4] Effect of length and targeting sequence variations on modified U1 snRNAs. Standard lentiviral vectors encoding GFP were produced in the absence (black bars) or presence of modified U1 snRNAs with targeting sequences to sites along the length of the 5' end of the vector genome vRNA molecule, including targeting lengths of 15 nucleotides (dark gray bars) or 9 nucleotides (light gray bars). The modified U1 snRNAs are named according to the first nucleotide of the targeting sequence site along the length of the 5' end of the vector genome vRNA molecule. The data bars for each modified U1 snRNA are placed below the approximate indicated position of each known functional sequence at the 5' end of the vector genome vRNA (not to scale). [Figure 5] Standard lentiviral vectors encoding GFP were produced in the absence (black bars) or presence of modified U1 snRNAs with targeting sequences to sites along the length of the 5' end of the vector genome vRNA molecule, including a targeting length of 15 nucleotides (dark gray bars). The modified U1 snRNAs are named according to the first nucleotide of the targeting sequence site along the length of the 5' end of the vector genome vRNA molecule. The data bars for each modified U1 snRNA are positioned below the approximate indicated position of each known functional sequence at the 5' end of the vector genome vRNA (not to scale). [Figure 6]Increased titer of lentiviral vectors encoding various transgenes by using modified U1 snRNA. Standard lentiviral vectors encoding GFP (pHIV-EF1a-GFP) or chimeric antigen receptors for CD19 (pHIV-EF1a-CD19) were produced in the absence (-) or presence of modified U1 snRNAs (256U1 or 305U1) targeting either site within the lentiviral vector packaging region or modified U1 snRNA targeting LacZ control (LacZU1). The modified U1 snRNAs are named according to the first nucleotide of the targeting sequence site within the lentiviral vector packaging region. The modified U1 snRNA expression constructs were supplied at two different doses: 1x and 4x. [Figure 7]The impact of aberrant splicing from the major splice donor site (MSD) in HIV-1-based lentiviral vectors. A. Schematic diagram showing the typical configuration of a third-generation (self-inactivating (SIN)) lentiviral vector expression cassette containing a functional major splice donor embedded within the stem-loop (SL2) of the packaging signal, and the types of mRNA produced during lentiviral vector production. Shown are the mRNA types produced from a "standard" lentiviral vector (LV) DNA cassette and (a) a lentiviral vector DNA cassette containing a functional mutation within the MSD region that suppresses or eliminates promiscuous activity from the MSD ("MSD-KO LV DNA cassette"). For both cassettes, full-length ("unspliced") vector RNA (vRNA) results from coexpression of rev, which is generally believed to bind to the rev response element (RRE) and suppress splicing from the MSD to splice acceptor 7 (sa7) (which is contained with the RRE sequence). In the case of standard lentiviral vector DNA cassettes, it is generally believed that in the absence of rev, splicing of all introns occurs efficiently ("spliced ​​out"). However, "aberrant" splice products can arise during lentiviral vector production. In this case, the MSD is very efficiently spliced ​​into splice acceptor or cryptic splice acceptor sites ("aberrant" splicing), typically "overlooking" the RRE-containing intron, and as a result, rev has minimal effect on this activity of the MSD. Lentiviral vector production can also be achieved by coexpressing modified U1 snRNA redirected to the packaging region of the MSD mutant lentiviral vector DNA cassette.(Legend: Pro, promoter; the region from 5'R to gag contains the packaging element {Ψ}; msd, major splice donor; cppt, central polypurine tract; Int, intron; sd / sa, splice donor / acceptor; GOI, gene of interest; gray arrows indicate the positions of the forward primer {f} and reverse primer {r} for assessing the proportion of unspliced ​​vRNA produced during third-generation lentiviral vector production. The post-transcriptional regulatory element {PRE} is not shown for clarity.) B. Standard third-generation lentiviral vector production was performed in HEK293T cells in the presence or absence of rev (+ / - rev), and total RNA was extracted from the cells after production. Total RNA was subjected to qPCR (SYBR Green) using two primer sets (positions labeled A) (f+rT amplified total transcripts produced from the lentiviral vector expression cassette; f+rUS amplified unspliced ​​transcripts). Therefore, the ratio of unspliced ​​vRNA transcripts to total vRNA transcripts was calculated and plotted. The data show that the ratio of unspliced ​​vRNA to total vRNA during the production of standard third-generation lentiviral vectors is moderate and varies depending on the internal transgene cassette (in this case, containing various promoters and the GFP gene). Furthermore, this ratio is only minimally increased by the action of rev. [Figure 8] An HIV-1 lentiviral vector genome containing three different promoter-GFP expression cassettes (EF1a, EFS, and CMV) was modified to functionally mutate the MSD, resulting in the "MSD-2KO" lentiviral vector genome or backbone (see Figure 15A for a description of the mutations). The vector was produced and titered in HEK293T cells using standard protocols. The data show that functional mutation of the MSD ("MSD-2KO") results in up to a 100-fold reduction in lentiviral vector titer. [Figure 9]Schematic diagram showing the construction of standard or MSD mutant lentiviral vector expression cassettes encoding the EF1a-GFP internal expression cassette and the type of mRNA produced during lentiviral vector production. (Legend: Pro, promoter; the region from 5'R to gag contains the packaging element {Ψ}; msd, major splice donor; cppt, central polypurine tract; Int, intron; sd / sa, splice donor / acceptor; GOI, gene of interest; gray arrows indicate the positions of the forward primer {f} and reverse primer {r} for assessing the proportion of unspliced ​​vRNA produced during third-generation lentiviral vector production. Post-transcriptional regulatory elements {PRE} are not shown for clarity.) B i. Standard lentiviral vectors or MSD-2KO lentiviral vectors were produced and titered in HEK293T cells in the presence or absence of tat, 179U1, or 305U1. ii. Total cytoplasmic mRNA was extracted from cells after production and analyzed by RT-PCR / gel electrophoresis using primers (f+rG) capable of detecting the major "aberrant" splice product from the SL2 splice region to the EF1a splice acceptor. The data show that modified U1 snRNA redirected to the 5' packaging region of the MSD-2KO lentiviral vector genome (vRNA) was able to increase the titer of both standard and MSD-2KO lentiviral vectors in a manner similar to tat. The MSD-2KO mutation prevented the detection of the "aberrant" splice product from the SL2 splice region to the EF1a splice acceptor (see Figure 9A). Importantly, the increase in titer with modified U1 snRNA was accompanied by the maintenance of virtually undetectable "aberrant" splice products, in contrast to the use of tat. [Figure 10]Standard lentiviral vectors or MSD mutant lentiviral vectors encoding a GFP internal cassette driven by the EF1a, EFS, or CMV promoter were produced and titered in HEK293T cells in the presence or absence of 256U1. The increase in lentiviral vector titer by using a modified U1 snRNA redirected to the 5' packaging region is independent of the promoter used within the transgene cassette. The data show that the attenuated phenotype of the MSD-2KO mutation is largely rescued by coexpression of the modified U1 snRNA, which therefore significantly increases the titer of the MSD mutant lentiviral vector genome disproportionately compared to the standard lentiviral vector genome. [Figure 11]The increase in titer of MSD-mutated lentiviral vectors using modified U1 snRNA redirected to the 5' packaging region is not related to the suppression of the potential activity of the 5' poly(A) signal within the 5' LTR. Previous reports have shown that MSD mutations activate the poly(A) signal within the 5'R sequence of the 5' LTR of the HIV-1 provirus and "mini-reporter" cassette, leading to premature termination of transcription, and binding of endogenous U1 snRNA and even redirected U1 snRNA can block this poly(A) activity. A. To assess the effects of two poly(A) signal mutants (pAM1 = AAUAAA > AACAAA; pAKO = deletion of AAUAAA) and the wild-type poly(A) signal (wt pA = AAUAAA) on transcriptional readthrough of the HIV-1 poly(A) site, we designed a GFP-poly(A) luciferase reporter cassette. Readthrough of the HIV-1 poly(A) signal was measured by luciferase activity normalized to GFP expression. B. To test whether modified U1 snRNAs acted similarly, a functional polyA mutation (pAm1) in the 5' polyA signal was introduced into MSD mutant lentiviral vector genomes containing EF1a-GFP or CMV-GFP expression cassettes. Standard and MSD mutant lentiviral vector genomes containing EF1a-GFP or CMV-GFP expression cassettes were also used. Lentiviral vectors were produced and titered in HEK293T cells in the presence or absence of 305U1. The data indicated that functional removal of the 5' polyA signal resulted in only a slight increase in lentiviral vector titer; therefore, the observed increase in lentiviral vector titer resulting from modified U1 snRNAs, particularly the MSD-2KO / polyA mutant lentiviral vector genome, may not be due to suppression of 5' polyA activity. [Figure 12]Several mutations were introduced into the 305U1 and 256U1 modified U1 snRNAs. These are known to eliminate the U1-70K protein binding to SL1, the U1A protein binding to SL2, or the Sm protein binding to or near SL4 of the vector genome. Standard or MSD-2KO lentiviral vectors encoding an EF1a-GFP internal cassette were produced and titered in the presence of these mutant modified U1 snRNAs, and titer values ​​were normalized to standard lentiviral vectors produced in the absence of modified U1 snRNA. The data indicate that the increase in MSD-2KO lentiviral vector titer by modified U1 snRNA is not dependent on U1-70K protein or U1A protein binding, but is dependent on the Sm protein binding site. Therefore, the increase in MSD-2KO lentiviral vector titer by using modified U1 snRNA redirected to the 5' packaging region is not related to any known function of U1 snRNA. [Figure 13] Increased titer of MSD mutant lentiviral vectors using modified U1 snRNA containing targeting sequences of various lengths. MSD-2KO lentiviral vectors containing an EF1a-GFP cassette were produced in HEK293T cells in the presence of modified U1 snRNA targeting the "305" region. In this case, each modified U1 snRNA contained a complementary retargeting sequence of various lengths. Increased titer was observed when modified U1 snRNA containing complementary lengths of 7 to 15 nucleotides was used, with the greatest effect observed at lengths of 10 nucleotides or more. [Figure 14]Maximum titer restoration / enhancement of MSD mutant lentiviral vectors is observed when modified U1 snRNA is targeted to the packaging region of the vector genome RNA. MSD-2KO lentiviral vectors containing an EF1-GFP cassette were produced in the presence of modified U1 snRNA with a targeting sequence to a site along the length of the 5' end of the vector genome vRNA molecule that contains a 15-nucleotide (or 9-nucleotide, where indicated) length of complementarity. The modified U1 snRNAs are named according to the first nucleotide of the targeting sequence site along the length of the 5' end of the vector genome vRNA molecule. The data bars for each modified U1 snRNA are positioned below the approximate indicated position of each known functional sequence at the 5' end of the vector genome vRNA (not to scale). [Figure 15]Illustration of functional major splice donor mutations, their effect on lentiviral vector titer, and restoration by modified U1 snRNA. A. The sequence of the stem-loop 2 (SL2) region of "wild-type" HIV-1 (NL4-3; the "canonical" sequence in this lentiviral vector genome) is shown at the top. This sequence contains the major splice donor site (MSD: consensus = CTGGT) and a cryptic splice donor site [used when the MSD site is mutated alone (crSD: consensus = TGAGT)]. Nucleotides at the splice position when the splice donor site is used are indicated in bold and with an arrow. Four functional MSD mutations have been described that eliminate both the MSD and crSD splicing activities: MSD-2KO, which mutates two "GT" motifs from the MSD and crSD sites (and is widely used in most examples); MSD-2KOv2, which also contains mutations that eliminate both the MSD and crSD sites; MSD-2KOm5, which introduces an entirely new stem-loop structure lacking the splice donor site; and ΔSL2, which completely deletes the SL2 sequence. The substitutions introduced into the SL2 sequence in the MSD-2KO, MSD-2KOv2, and MSD-2KOm5 mutations are indicated in lowercase italics. B. Four lentiviral vector genome variants containing functional MSD mutations (as shown in Figure 15A) were cloned with an EFS-GFP internal cassette, and the MSD-2KO or MSD-2KOm5 variants were further cloned with an EF1a-, CMV-, or huPGK-GFP internal cassette. Standard and MSD-mutated LVs were produced and titered in HEK293T cells in the presence or absence of 256U1. The data indicate that the degree of attenuation of lentiviral vector titer can vary depending on the specific mutation, and that the MSD-2KOm5 mutant generally produced a less attenuated phenotype. When coexpressed during production, modified U1 snRNA was able to increase lentiviral vector titers for four lentiviral vector genome variants containing functional MSD mutations.The increase in titer was greatest when 256U1 was expressed together with the MSD-mutated LV genome containing the MSD-2KOm5 sequence. [Figure 16]The modified U1 snRNA expression cassette can be placed in the lentiviral vector genome plasmid backbone for ease of use in transient transfection protocols. Many of the examples use a separate modified U1 snRNA expression plasmid for co-transfection with the lentiviral vector component plasmids in the production of lentiviral vectors. To identify "permissive" sites on the lentiviral vector genome plasmid backbone that allow for the provision of the modified U1 snRNA cassette in cis during transient transfection, three variants were cloned. A. Schematic diagram of lentiviral vector genome variants that provide the modified U1 snRNA cassette in cis during transient transfection. Version 1 ("[cis]ver1") and version 3 ("[cis]ver3") place the modified U1 snRNA cassette between the resistance marker and the origin of replication. In this case, the modified U1 snRNA cassette is inverted relative to the lentiviral vector genome cassette (the orientation of the resistance marker is different between ver1 and ver3). Version 2 ("[cis]ver2") placed a modified U1 snRNA cassette upstream of the lentiviral vector genome cassette in the same orientation. (Legend: Pro, promoter; the region from 5'R to gag contains the packaging element {Ψ}; msd, major splice donor {shown here as MSD-2KO}; RRE, rev response element; cppt, central polypurine tract; Transgenic, heterologous sequence containing the therapeutic payload; U1-Pro, U1 promoter; Term[3'box], U1 transcription terminator.) B. Three "cis" versions of the MSD-2KO lentiviral vector genome plasmid containing the EF1a-GFP cassette were used to produce lentiviral vectors in HEK293T cells in parallel with the "trans" approach.In the "trans" approach, the same MSD-2KO lentiviral vector genome (without the modified U1 snRNA cassette inserted into the backbone) was produced in the presence or absence of modified U1 snRNA supplied by co-transfection with a separate plasmid. The data demonstrate that the use of a "cis" lentiviral vector genome, similar to co-transfection of a separate modified U1 snRNA-encoding plasmid, can increase MSD-2KO lentiviral vector titers. [Figure 17] Further demonstration of the use of modified U1 snRNA to increase the titer of standard lentiviral vectors encoding therapeutic transgenes. Standard lentiviral vectors containing EF1a-driven transgene cassettes encoding codon-optimized or wild-type human alpha-1-antitrypsin fused to GFP via the T2A peptide or encoding a chimeric antigen receptor (CAR) for the cancer antigen 5T4 were produced in serum-free suspension HEK293T cells in the presence or absence of modified U1 snRNA (256U1) and titered by integration assay and GFP-FACS assay (where indicated). The data show that vector titer increased approximately threefold when modified U1 snRNA was provided during production. [Figure 18] The successful isolation of HEK293T cells stably expressing modified U1 snRNA, enabling propagation of standard or MSD-2KO lentiviral vectors, demonstrates that modified U1 snRNA cassettes can be introduced into lentiviral vector packaging and producer cell lines. Standard or MSD-2KO lentiviral vector genomes containing the EFS-GFP cassette were produced in HEK293T or HEK293T.305U1 (9 nt mutant) cells with or without the additional 305U1 plasmid. The data demonstrate that stable cassettes expressing modified U1 snRNA can be introduced into cells without toxicity. [Figure 19]During lentiviral vector production, aberrantly spliced ​​mRNAs expressing transgenes are blocked in the MSD-2KO lentiviral vector, reducing the amount of transgene mRNA that needs to be targeted by TRAP when using the TRiP system. A. Schematic diagram of the "TRiP" lentiviral vector genome encoding the EF1a-GFP transgene cassette. Here, a TRAP binding site (tbs) is located within the 5'UTR of the cassette (providing TRAP during vector production reduces transgene expression levels). During MSD-2KO lentiviral vector production, full-length unspliced ​​packageable vRNA and transgene mRNA are the major forms of RNA produced from the lentiviral vector cassette (i) (if the transgene promoter is active during production). However, promiscuous activity of MSD in standard lentiviral vector genomes results in additional "aberrant" splice products that may encode the transgene (ii). This can occur independently of the internal transgene promoter, i.e., a tissue-specific promoter. (Legend: Pro, promoter; the region from 5'R to gag contains the packaging element {Ψ}; msd, major splice donor; cppt, central polypurine tract; Int, intron; sd / sa, splice donor / acceptor; GOI, gene of interest; gray arrows indicate the location of the forward primer {f} and reverse primer {r} for assessing the proportion of unspliced ​​vRNA produced during third-generation lentiviral vector production. The post-transcriptional regulatory element {PRE} is not shown for clarity.) B. Standard or MSD-2KO lentiviral vector genome plasmids containing the EF1a-GFP cassette were used to produce lentiviral vectors in HEK293T cells, and GFP expression scores were obtained (% GFP x MFI). Compared to the total amount of GFP produced in culture during standard lentiviral vector production, MSD-2KO showed a significant effect of reducing the amount of GFP produced, even in the absence of TRAP. Thus, the inhibitory effect of TRAP was enhanced by the use of the MSD-2KO lentiviral vector genome, significantly reducing the levels of GFP in the cultures. [Figure 20] The increase in lentiviral vector titer with the use of modified U1 snRNA correlates with increased vector RNA packaging into virions. Total RNA was extracted from RNase-treated crude vector supernatants of the vectors produced in Example 5 (see Figure 6). Purified RNA was subjected to RT-qPCR using primers directed against the HIV-1 packaging signal in the vector genome RNA. The increase in vRNA signal in crude vector harvests produced in the presence of modified U1 snRNA relative to vRNA was similar in magnitude to the increase in vector titer shown in Figure 6. [Figure 21] The use of modified U1 snRNA increased the titer of α1-antitrypsin (α1AT)-encoding VSVG pseudotyped lentiviral vectors and concomitantly suppressed transgene expression during production. HIV-1-based LVs encoding codon-optimized (co) or wild-type (wt) α1AT proteins translationally linked to GFP expression (via the T2A peptide) were produced in serum-free HEK293T suspension cells in the absence or presence of 256U1. [A] Adherent HEK293T cells were transduced and clarified vector harvests were titered by flow cytometry. [B] Post-production cell lysates were immunoblotted for transgene protein and β-actin. A slight but observable decrease in transgene expression was evident in cells cotransfected with the 256U1 expression plasmid. [Figure 22]The use of modified U1 snRNA increased the titer of α1-antitrypsin (α1AT)-encoding Sendai virus (SeV) envelope pseudotyped lentiviral vectors and concomitantly suppressed transgene expression during production. HIV-1-based LVs encoding the α1AT protein translationally linked to GFP expression (via the T2A peptide) were produced in adherent or suspension (serum-free) HEK293T cells in the absence or presence of 256U1. SeV-F / HN pseudotyped LVs required activation by Trispin treatment before transduction. [A] Adherent HEK293T cells were transduced and the clarified vector harvest was titered by flow cytometry. [B] Post-production cell lysates were immunoblotted for transgene protein and β-actin. A slight but observable decrease in transgene expression was evident in cells cotransfected with the 256U1 expression plasmid. The increase in LV titer mediated by 256U1 was more pronounced with SeV F / HN-pseudotyped vectors than with VSVG-pseudotyped vectors due to the decrease in α1-antitrypsin expressed during production (Fig. 21). The SeV F protein requires activation by trypsin before transduction, and therefore, α1AT in the crude vector likely inhibits this activation step. [Figure 23] Use of modified U1 snRNA to increase the titer of lentiviral vectors containing an inverted transgene cassette. [A] Schematic diagram of the LV genome expression cassette encoding an inverted β-globin gene (containing the necessary exons / introns for efficient expression in primary cells) driven by the LCR-β-globin promoter. [B] LV preparations were produced in suspension (serum-free) HEK293T cells in the absence or presence of the indicated modified U1 snRNA. Clarified vector harvests were titered by transduction of adherent HEK293T cells and subsequent integration assays. [Figure 24]Increased lentiviral vector titer with the use of modified U1 snRNA is observed not only in crude vector material but also in concentrated vector material. HIV-1-based lentiviral vectors encoding an EF1a promoter-driven firefly luciferase / GFP dual reporter cassette were produced in suspension (serum-free) HEK293T cells in the absence or presence of 256U1. The majority of the clarified vector harvest was concentrated by centrifugation to obtain an approximately 20-fold concentration factor. The clarified vector harvest and concentrated vector were titered by transduction of adherent HEK293T cells followed by flow cytometry to measure GFP-positive cells. [Figure 25]Development of a TaqMan-based RT-qPCR assay for the detection of modified U1 snRNA to assess expression levels and residues. [A] Schematic diagram showing the 256 U1 snRNA molecule and the binding positions of the forward / reverse primers and FAM / TAMRA-conjugated probe. The main difference between endogenous U1 snRNA and the modified U1 snRNA described in this invention is the 5' end of the molecule, where the natural splice donor annealing sequence is replaced by a targeting sequence, allowing the modified U1 snRNA to anneal to the vector genome RNA. Due to the requirement for a cDNA synthesis step using a reverse primer (using reverse transcriptase), both endogenous and modified U1 snRNA contribute to the pool of cDNA generated during cDNA synthesis of cellular RNA and (potentially) RNA extracted from vector particles. Therefore, to distinguish between endogenous and modified U1 snRNA, a forward primer is designed to anneal to the vRNA targeting sequence at the 5' end. [B] Plotted Ct thresholds obtained from Taqman qPCR (+ / - RT step) of total RNA extracted from cell lysates obtained from transfections with or without p256U1. A standard curve (filled circles) was generated from p256U1 samples (2 × 10 copies per reaction, 10-fold serial dilutions) and showed good range and linearity. The approximately 2-fold cycle difference between untransfected cell samples and p256U1-transfected cells (no RT step) indicates the possibility of residual p256U1 DNA levels associated with the samples, even though the samples were treated with DNase after the RT step. The average difference in Ct between +RT- and -RT-treated samples from p256U1-transfected cells was 19.4 cycles; at an 80-fold dilution of the sample (i.e., approximately 10 copies per reaction), this difference was 20 cycles. [Figure 26]Dose response of adding a modified U1 snRNA expression plasmid to the transfection mixture during the transient transfection method for lentiviral vector production. The HIV-1-based LV-CAR vector (encoding an EF1a promoter-driven cassette expressing a CD19-targeting CAR) was produced in suspension (serum-free) HEK293T cells by transient transfection in the absence or presence of increasing amounts of cotransfected p256U1. [A] Post-production cells were subjected to total RNA extraction, and levels of vector genomic RNA (vRNA) or 256U1 snRNA were quantified by RT-qPCR. All data were normalized based on control RT-qPCR performed against the endogenous transcript (RPH1). [B] Clarified LV-CAR vector supernatant was titered by transduction of adherent HEK293T cells followed by integration assay (qPCR of extracted host cell DNA using vRNA primers). The data show a correlation between 256U1 snRNA and vRNA levels in producer cells, leading to a similar increase in output vector titer. [Figure 27]An example of the application of polydispersity modeling (via "design of experiments" [DoE]) to optimize the ratio of modified U1 snRNA to LV component plasmid for the production of lentiviral vectors encoding therapeutic transgenes. The HIV-EF1a-5T4CAR vector was produced in suspension (serum-free) HEK293T cells at a 40 mL shake flask scale. Input levels of GagPol and rev were fixed, while levels of genome, VSVG, and 256U1 plasmid were varied. The clarified crude recovered vector was titered by transduction of adherent HEK293T cells followed by immunoflow cytometry using an anti-CAR antibody (light gray bars; "Test" T1-28). Titer values ​​obtained from the DoE experiment allowed prediction of the optimal level of p256U1 input. Further vector preparations were then produced either at this input level ("DoE prediction" D1-3) or in the absence of p256U1 ("Platform" P1-3). This optimization experiment allowed the application of p256U1, resulting in an approximately 10-fold increase in HIV-EF1a-5T4CAR production titer. [Figure 28] Production and enrichment of HIV-EF1a-5T4CAR vector in the presence or absence of 256U1 to produce concentrated vector for transduction of primary T cells and analysis of residual 256U1 snRNA. HIV-EF1a-5T4CAR ("LV-CAR") vector was produced by transient transfection of suspension (serum-free) HEK293T cells at a 250 mL shake flask scale and subjected to ion exchange chromatography, DNase treatment with salt-active nuclease (SAN), and subsequent low-speed centrifugation. The vector preparation was titered by transduction of HEK293T cells and subsequent integration assay. [Figure 29]Detection and quantification of residual 256U1 snRNA in vector preparations. Vector samples from the production of HIV-EF1a-5T4CAR ("LV-CAR") produced in the presence or absence of 256U1 (see Figure 28) were subjected to total RNA extraction followed by RT-qPCR analysis of vector-associated RNA to quantify vRNA and 256U1 residual DNA levels / ratios. Data indicate that the initially high levels of 256U1 snRNA detected in the clarified vector harvest were primarily due to "free" 256U1 snRNA, which could be removed by treatment with benzonase (benzonase was not used to treat the clarified vector harvest during purification). This treatment reduced the 256U1 to vRNA ratio to 1:20, which was further reduced to 1:32 after salt-active nuclease (SAN) treatment during downstream processing / concentration. [Figure 30]Comparative protein analysis by mass spectrometry of enriched / purified preparations of HIV-EF1a-5T4CAR produced in the presence or absence of 256U1. Enriched / purified LV-CAR preparations (see Figures 28 and 29) produced to transduce primary T cells (see Table IV) were analyzed by mass spectrometry to assess major differences in protein content that may arise from the expression of 256U1-modified U1 snRNA molecules during LV production. The top 400 protein hits for the vector produced in the presence of 256U1 (LV-CAR[+256U1]) were ranked 1–400 based on their relative abundance as a proportion of the total, and the relative abundance of these hits in LV-CAR or LV-CAR[+256U1] was plotted on the y-axis. Of these 400 proteins, the top 100 constituted approximately 70% of the total protein abundance, and the top 10 constituted approximately 30%. The top two most frequent hits were Gag and VSV-G, both from LV preparations; other cellular factors known to be incorporated at high levels into HIV-1 virions include basigin, HSPc-71K, agrin, and cyclophilin A, the latter of which specifically binds to the capsid. This comparison showed little difference in the protein composition of the two LV preparations. The ratio of the abundance of peptides mapping to Gag and Pol (Gag vs. Pol) was approximately 16 for both LV preparations, consistent with the expected ratio of approximately 20 for HIV-1, indicating the good quality of the data. [Figure 31]Generation of CAR-T cells using the HIV-EF1a-5T4CAR vector manufactured with or without 256U1. Approximately 1.5 × 10 peripheral blood mononuclear cells (PBMCs) from three healthy donors were cultured in the presence of CD3 / CD28 T cell expander beads and incubated with IL-2. Activated T cells were transduced with the enriched vector samples "LV-CAR" and "LV-CAR[+256U1]" (see Figures 28-30 and Table IV) at an MOI of 1.25, and in the case of LV-CAR[+256U1], at an MOI of 0.3. [A] Total viable cell counts were monitored up to day 13 (D13) post-transduction, after which a frozen viable cell bank (1 × 10 vc / vial) was generated. CAR-T cells were revived and expanded for 5 days (R+5) and ready for cell killing and cytokine release assays (see Figures 32 and 33). [B] Transduction rates were measured on day 8 post-transduction (D8) and upon recovery from frozen stocks. [Figure 32] Functionality of CAR-T cells generated using LV-CAR vectors produced with or without 256U1 was assessed; cytokine release was assessed. The recovered CAR-T cells were expanded for an additional 5 days. All viabilities exceeded 97%. Approximately 1 x 105 CAR-T cells were co-cultured with an equal number of target cell lines: THP-1, Kasumi-1, and SKOV-3 (all 5T4-positive), and AML-193 (a 5T4-negative cell line). After 24 hours, culture supernatants were analyzed for granzyme B [A] and interferon-γ activity [B] using cytometric beads. [Figure 33]Functionality of CAR-T cells generated using LV-CAR vectors produced with or without 256U1 was assessed for target cell killing. The recovered CAR-T cells were expanded for an additional 5 days. All viabilities exceeded 97%. Approximately 1 x 105 CAR-T cells were co-cultured with an equal number of target cell lines: THP-1, Kasumi-1, and SKOV-3 (all 5T4-positive), and AML-193 (a 5T4-negative cell line). Target cells were labeled with a fluorescent cell-tracking dye to enable subsequent identification by flow cytometry. After 40 hours, cells were harvested and stained with a fluorescent viability dye. The percentage of nonviable target cells in each experimental well was measured by flow cytometry and compared with the viability of target cell-only cultures (no CAR-T cells added). [Figure 34] Analysis of residual vector-associated RNA in CAR-T growth cultures after transduction with HIV-EF1a-5T4CAR manufactured in the presence of 256U1. During CAR-T cell expansion after transduction with HIV-EF1a-5T4CAR (+256U1; see Figure 31) at two different MOIs, cell pellets were collected on days 8 and 13. Total RNA was extracted and RT-qPCR was performed for RPH1 mRNA, vRNA (Psi), and 256U1 snRNA. The difference between the abundance of endogenous RPH1 transcripts and the abundance of residual 256U1 snRNA was calculated by the delta Ct method. [Figure 35] HIV-EF1a-CAR(CD19) vector production by transient transfection of lentiviral vector packaging cell lines with or without p256U1 in shake flasks and bioreactors. Either the HIV-EF1a-CAR_CD19 or HIV-EF1a-CAR_CD19-T2A-GFP genomic plasmid was transfected into suspension serum-free adapted lentiviral vector packaging cell lines (PACs) in the presence or absence of p256U1. Production was performed in either 40 mL shake flasks or 250 mL bioreactors. Suspension serum-free adapted HEK293T cells served as a control, in which all vector component plasmid DNAs were also cotransfected. [Figure 36] Enhanced lentiviral vector production from a suspension (serum-free) adapted HEK293T cell line stably transfected with a 256U1 snRNA expression cassette. The suspension (serum-free) adapted HEK293T cell line, "256U1c39," was isolated from HEK293T cells stably transfected with a 256U1 snRNA expression cassette operably linked to a hygromycin B resistance marker cassette. The increase in HIV-EF1a-5T4CAR titer compared to the parental HEK293T cell line was assessed over a 10-week period in the presence or absence of selective pressure. The data show that the 256U1c39 clone stably produced 256U1 snRNA at levels close to or equal to those of the transiently transfected HEK293T parental cells. [Figure 37] Testing the length of the modified U1 snRNA retargeting sequence. HIV-EF1a-GFP, HIV-EF1a-CARCD19, or HIV-EF1a-5T4CAR vectors were produced in suspension (serum-free) HEK293T cells in the presence of modified U1 snRNA targeting position 305 within the LV packaging region of the vRNA. Each 305U1 variant tested contained targeting sequences of various lengths, ranging from 5, 7, 9, to 15 nucleotides. The variants were compared with unmodified U1 and 256U1 (15 nt). Clarified vector supernatants were titered by transduction of adherent HEK293T cells followed by flow cytometry [A] or integration assay [B]. [Figure 38]The increase in lentiviral vector titer with modified U1 snRNA appears to be unrelated to the reported ability of the "AU" dinucleotide dependency of U1 snRNA in generating splicing-involved complexes. HIV-EF1a-GFP vectors were produced in suspension (serum-free) HEK293T cells in the presence of the indicated dinucleotide mutants of 256U1 snRNA (see Table V) and titered on adherent HEK293T cells. Relative titers (vs. those in the absence of 256U1) were plotted and ranked from highest to lowest predicted CBP20 binding score and impact on titer (13nt mutant, light gray bars) according to Yeh et al. (2017). The dotted line indicates the titer increase with the 256U1_13_aT control mutant. There did not appear to be a correlation between the predicted ability of each dinucleotide mutant's CAP binding score and increased vector titer. [Figure 39] Fine-tuning the modified U1 targeting site. Based on the apparent "hot spot" identified by the 256 U1 snRNA, modified U1 snRNAs containing a target annealing length of 13 nucleotides were designed (Table VI) (see Figures 4 and 5). These were designed to shift the target site in approximately 2-nt increments upstream or downstream of the nt 256 target site in the HIV-1-based LV genome. HIV-EF1a-GFP vectors were produced in suspension (serum-free) HEK293T cells by transient transfection in the absence or presence of each of the indicated mutant modified U1 snRNAs. Clarified vector supernatants were titered by transduction of adherent HEK293T cells followed by analysis by flow cytometry. [Figure 40]Increased titer of EIAV-based lentiviral vectors by cotransfection of producer cells with modified U1 snRNA directed against the 5' packaging region of EIAV vRNA. Suspension (serum-free) HEK293T cells were transfected with either pEIAV-CMV-GFP or pEIAV-EF1a-GFP genomic plasmid, as well as pGagPol, pRev, and pVSVG, in the presence or absence of the indicated modified U1 snRNA expression plasmids (see Table VI). Clarified vector supernatants were titered by transduction of adherent HEK293T cells followed by flow cytometry. Relative titers are plotted compared to vectors produced in the absence of the modified U1 snRNA expression plasmid (striped bars). [Figure 41] Increased titer of SIVagm-based lentiviral vectors by cotransfection of producer cells with modified U1 snRNA directed against the 5' packaging region of SIVagm vRNA. Suspension (serum-free) HEK293T cells were transfected with SIV vector components in the presence or absence of the indicated modified U1 snRNA expression plasmids (see Table VIII). Clarified vector supernatants were titered by transduction of adherent HEK293T cells and subsequent integration assays.

[0079] Detailed Description of the Invention General definition The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology and immunology which are within the capabilities of those skilled in the art and are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13, and 16, John Wiley & Sons, New York, NY; B. Roe, J. Crabtree, and A. Kahn (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; M. J. Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and D. M. J. Lilley and J. E. Ahlberg (1992) Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press. Each of these general texts is incorporated herein by reference.

[0080] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise specified, "rev" and "gag-pol" refer to proteins and / or genes of a lentiviral vector.

[0081] As used herein, the term "protein" includes proteins, polypeptides, and peptides. As used herein, the term "protein" includes single polypeptide molecules and complexes of multiple polypeptides in which the individual constituent polypeptides are linked by covalent or non-covalent means. As used herein, the terms "polypeptide" and "peptide" refer to polymers in which amino acid monomers are linked together by peptide or disulfide bonds.

[0082] As used herein, the term "amino acid sequence" is synonymous with the terms "polypeptide" and / or "protein." In some instances, the term "amino acid sequence" is synonymous with the term "peptide." In some instances, the term "amino acid sequence" is synonymous with the term "enzyme."

[0083] As used herein, the term "nucleotide sequence" is synonymous with the terms "polynucleotide" and / or "nucleic acid sequence."

[0084] The present disclosure is not limited by the exemplary methods and materials disclosed herein; any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. Numeric ranges are inclusive of the values ​​defining the range. Unless otherwise indicated, all nucleic acid sequences are presented left to right in 5' to 3' orientation, respectively, and amino acid sequences are presented left to right in amino to carboxy orientation, respectively.

[0085] Where a range of values ​​is disclosed, unless the context clearly contradicts otherwise, each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range is also understood to be specifically disclosed. Each smaller range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is included within the disclosure. The upper and lower limits of these smaller ranges may independently be included in or excluded from the range, and, in the case of any specifically excluded limits in a stated range, each range in which either or both limits are included in the smaller range is also included in the disclosure. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0086] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0087] As used herein, the words "comprise," "including," and "including" are synonymous with "include," "comprise," or "contain," and are inclusive or open-ended and do not exclude additional, non-recited members, elements, or method steps. The words "comprise," "comprise," and "comprising" also include the word "consisting of."

[0088] Modified U1 snRNA The present inventors have surprisingly found that the output titer of lentiviral vectors can be increased by co-expressing a non-coding RNA based on U1 snRNA that has been modified so that it no longer targets the endogenous sequence (splice donor site) but now targets a sequence within the vRNA molecule. The present invention relates to such modified U1 snRNA and a novel method for increasing the production titer of lentiviral vectors. This approach consists of co-expressing the modified U1 snRNA with other vector components during vector production. The modified U1 snRNA is designed such that binding to the consensus splice donor site is eliminated by replacing the consensus splice donor site with a heterologous sequence complementary to the target sequence within the vector genome vRNA. The present invention describes optimal features and various modes of application of the modified U1 snRNA, including the length, design, and expression modes of the target sequence and complementarity.

[0089] Human U1 snRNA (small nuclear RNA) is 164 nt long and has a distinct structure consisting of four stem-loops (see Figure 1). U1 snRNA, an endogenous noncoding RNA, binds to a consensus splice donor site (e.g., 5'-MAGGURR-3'; where M is A or C and R is A or G) via a natural splice donor annealing sequence (e.g., 5'-ACUUACCUG-3') during the early stages of intron splicing. Stem-loop I binds to the U1A-70K protein, which has been shown to be important for poly(A) repression. Stem-loop II binds to the U1A protein, and the 5'-AUUUGUGG-3' sequence binds to the Sm protein, which, together with stem-loop IV, is important for U1 snRNA processing. In the present invention, the modified U1 snRNA is modified to introduce a heterologous sequence complementary to a target sequence within the vector genome vRNA molecule at the site of the natural splice donor targeting sequence (see Figure 1).

[0090] As used herein, the terms "modified U1 snRNA," "redirected U1 snRNA," "retargeted U1 snRNA," "recycled U1 snRNA," and "mutant U1 snRNA" refer to a U1 snRNA that has been modified so that it no longer binds to the consensus 5' splice donor site sequence (e.g., 5'-MAGGURR-3') that it uses to initiate the splicing process of a target gene. Thus, a modified U1 snRNA is a U1 snRNA that has been modified so that it no longer binds to a splice donor site sequence (e.g., 5'-MAGGURR-3') based on the complementarity of the donor site sequence with the natural splice donor annealing sequence at the 5' end of the U1 snRNA. Instead, the modified U1 snRNA is designed to bind to a nucleotide sequence with a unique RNA sequence (target site) within the packaging region of a lentiviral vector genome molecule, a sequence unrelated to gene splicing. The nucleotide sequence within the packaging region of a lentiviral vector genome molecule can be preselected. Thus, the modified U1 snRNA is a U1 snRNA whose 5' end has been modified to bind to a nucleotide sequence within the packaging region of a lentiviral vector genome molecule, and as a result, the modified U1 snRNA binds to the target site sequence based on the complementarity of the target site sequence with the short sequence at the 5' end of the modified U1 snRNA.

[0091] The 5' packaging region of a lentiviral vector can have a sequence known in the art. For example, the 5' packaging region of a lentiviral vector can be any of the following: [ka] TIFF0007787064000002.tif222165TIFF0007787064000003.tif241164TIFF0007787064000004.tif181164

[0092] Appropriate modified U1 snRNAs can be designed to bind to the packaging region of a particular vector type. For example, the use of modified U1 snRNAs to increase the production titer of lentiviral vectors can be achieved by following the following general procedure.

[0093] 1. The lentiviral vector genome sequence should be obtained and a set of target sequences should be identified within the 5' packaging sequence region of the vector genome RNA molecule. The extended 5' packaging sequence region extends from the first nucleotide of the vector genome RNA molecule to the 3' nucleotide of the remaining wild-type gag sequence (which is typically retained as part of the packaging sequence).

[0094] 2. For initial target screening, we recommend identifying a panel of 15-nucleotide-long target sequences, initially identifying 15–20 distinct (non-overlapping) sequences, evenly distributed across the packaging sequence from the first nucleotide of the vRNA to approximately 50 nucleotides of the gag region, with fewer sequences identified within the retained gag region.

[0095] 3. These target sequences present in the vector genome RNA (5'-3') are reverse-complemented to the 15-nucleotide sequence encoded within the first several nucleotides of the modified U1 snRNA molecule (5'-3'). Target Annealing You should get an array.

[0096] 4. Insert the target annealing sequence into the U1 snRNA expression cassette (containing the U1 promoter and termination region) to replace native U1 snRNA nucleotides 3-11. That is, the "AT" dinucleotide (nucleotides 1 and 2 of native U1 snRNA ["AU" in the snRNA molecule]) should be retained upstream of the target annealing sequence, where "A" is the transcription start site. This can be accomplished by standard molecular cloning / gene synthesis techniques.

[0097] 5. The panel of modified U1 snRNA expression constructs should then be screened by producing lentiviral vectors encoding the transgenic sequences of interest, where each modified U1 snRNA expression construct is expressed individually with the vector components. This can be most conveniently done by transient co-transfection with the vector components, but can also be done in a packaging or producer cell line. The resulting vector supernatants are then titered to empirically determine the primary target region that results in the greatest titer increase.

[0098] 6. The modified U1 snRNA can be further improved by generating mutants containing target annealing sequences that incrementally target the vector genome RNA upstream and downstream of the initial empirically identified target site. This incremental scanning can be achieved by incrementally shifting the target annealing sequence by one, two, three, or four or more nucleotides per mutant, respectively, upstream or downstream of the initial empirically identified target site, optionally continuing to the target position tested in the previous (initial) screen. This can identify the optimal target site within the vector genome RNA.

[0099] 7. The modified U1 snRNA can be further improved by generating variants containing target annealing sequences of different lengths. It is recommended to take the modified U1 snRNA identified from a previous screen (which may have a 15-nucleotide target annealing sequence) and design variants with stepwise shortening or lengthening of the target annealing sequence. In this case, a new panel of variants is generated, where the target annealing sequence of each variant can be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides in length. This new panel of modified U1 snRNA variants can then be screened as before.

[0100] 8. The modified U1 snRNA can be further improved by generating variants containing alternative nucleotides at positions 1 and 2. Specifically, the "AT" dinucleotide ("AU" in the snRNA molecule) can be altered to deviate from this consensus, e.g., to "GA." This new panel of modified U1 snRNA variants can then be screened as before.

[0101] 9. The modified U1 snRNA expression construct can be encoded within a DNA molecule separate from the lentiviral vector components (e.g., plasmid DNA), or can be operably linked to DNA encoding the vector genome or gagpol or other DNA components that are co-transfected with the vector components (e.g., a TRAP-expressing plasmid).

[0102] 10. The modified U1 snRNA expression construct can be stably transfected to generate a cell line, which can be used to produce a lentiviral vector by transient transfection. For example, the cell line can be stably transfected with a transcriptional repressor such as tetR and / or a translational repressor such as TRAP, or both types of expression control proteins.

[0103] 11. The modified U1 snRNA expression construct can be stably transfected with other lentiviral vector components to generate packaging or producer cell lines.

[0104] 12. Another method for obtaining such stable cell lines is to insert a modified U1 snRNA expression cassette into a self-inactivating retroviral or lentiviral vector, produce vector virions encoding the modified U1 snRNA expression cassette, and transduce cells at a controlled multiplicity to isolate stable cell lines that are more likely to contain the desired copy number of the modified U1 snRNA expression cassette. The self-inactivating retroviral or lentiviral vector may also contain a selectable marker cassette.

[0105] 13. To assess the RNA or DNA copy number of modified U1 snRNA sequences in lentiviral vector production cells or lentiviral vector products, an RT-qPCR assay can be developed in which a forward primer anneals to a specific target annealing sequence in the modified U1 snRNA to specifically detect the modified U1 snRNA over the endogenous / native U1 snRNA.

[0106] As used herein, the terms "native splice donor annealing sequence" and "native splice donor targeting sequence" refer to a short sequence at the 5' end of endogenous U1 snRNA that is broadly complementary to the consensus 5' splice donor site of an intron. The natural splice donor annealing sequence can be 5'-ACUUACCUG-3'.

[0107] As used herein, the term "consensus 5' splice donor site" refers to a consensus RNA sequence at the 5' end of an intron used in splice site selection, for example having the sequence 5'-MAGGURR-3'.

[0108] As used herein, the terms "nucleotide sequence within the packaging region of a lentiviral vector genome sequence," "target sequence," and "target site" refer to a site having a specific RNA sequence within the packaging region of a lentiviral vector genome molecule that has been preselected as a target site for binding to modified U1 snRNA.

[0109] As used herein, the terms "packaging region of a lentiviral vector genome molecule" and "packaging region of a lentiviral vector genome sequence" refer to the region at the 5' end of a lentiviral vector genome from the beginning of the 5' U5 domain to the end of the gag gene-derived sequence. Thus, the packaging region of a lentiviral vector genome molecule includes the 5' U5 domain, the PBS element, the stem-loop (SL)1 element, the SL2 element, the SL3ψ element, the SL4 element, and the gag gene-derived sequence. It is common in the art to provide a complete gag gene in trans to the genome during lentiviral vector production to enable the production of replication-deficient viral vector particles. The nucleotide sequence of the gag gene provided in trans need not be encoded by wild-type nucleotides but can be codon-optimized. Importantly, the primary characteristic of the gag gene provided in trans is that it encodes and directs the expression of gag and gagpol proteins. Therefore, when the complete gag gene is provided in trans during lentiviral vector production, it will be understood by those skilled in the art that the term "packaging region of a lentiviral vector genome molecule" can refer to the region at the 5' end of the lentiviral vector genome molecule from the start of the 5' U5 domain to the "core" packaging signal in the SL3 ψ element, and the native gag nucleotide sequence from the ATG codon (present in SL4) to the end of the remaining gag nucleotide sequence present on the vector genome.

[0110] As used herein, the term "gag gene-derived sequence" refers to any naturally occurring sequence of the gag gene derived from the ATG codon through nucleotide 688 (Kharytonchyk, S. et al., 2018, J. Mol. Biol., 430:2066-79) that may be present (e.g., remain) within the vector genome.

[0111] As used herein, the terms "introducing a heterologous sequence within the first 11 nucleotides of U1 snRNA, which contains the natural splice donor annealing sequence," "introducing the heterologous sequence within the 9 nucleotides 3-11," and "introducing a heterologous sequence within the first 11 nucleotides at the 5' end of U1 snRNA" include replacing, in whole or in part, the first 11 nucleotides or the 9 nucleotides 3-11 of U1 snRNA with the heterologous sequence, or modifying the first 11 nucleotides or the 9 nucleotides 3-11 of U1 snRNA so that they have the same sequence as the heterologous sequence.

[0112] As used herein, the terms "introducing a heterologous sequence into the native splice donor annealing sequence" and "introducing a heterologous sequence into the native splice donor annealing sequence at the 5' end of U1 snRNA" include replacing the native splice donor annealing sequence, in whole or in part, with the heterologous sequence, or modifying the native splice donor annealing sequence so that it has the same sequence as the heterologous sequence.

[0113] As used herein, the term "enhancing lentiviral vector titer" includes "increasing lentiviral vector titer" and "improving lentiviral vector titer."

[0114] Thus, in one aspect, the present invention provides a modified U1 snRNA that has been modified to bind to a nucleotide sequence within the packaging region of a lentiviral vector genome sequence.

[0115] In some embodiments, the modified U1 snRNA is modified at the 5' end relative to the endogenous U1 snRNA to introduce a heterologous sequence complementary to a nucleotide sequence within the packaging region of the lentiviral vector genome sequence.

[0116] In some embodiments, the modified U1 snRNA is modified at the 5' end relative to the endogenous U1 snRNA to introduce a heterologous sequence into the natural splice donor annealing sequence that is complementary to a nucleotide sequence within the packaging region of the lentiviral vector genome sequence.

[0117] The modified U1 snRNA may be modified at the 5' end relative to the endogenous U1 snRNA so as to replace the sequence containing the natural splice donor annealing sequence with a heterologous sequence complementary to the nucleotide sequence.

[0118] The modified U1 snRNA can be a modified U1 snRNA variant. The U1 snRNA variant modified according to the present invention can be a naturally occurring U1 snRNA variant, a U1 snRNA variant containing a mutation in the stem-loop I region that eliminates U1-70K protein binding, or a U1 snRNA variant containing a mutation in the stem-loop II region that eliminates U1A protein binding. The U1 snRNA variant containing a mutation in the stem-loop I region that eliminates U1-70K protein binding can be U1_m1 or U1_m2, preferably U1A_m1 or U1A_m2.

[0119] In some embodiments, a modified U1 snRNA of the invention comprises a nucleotide sequence having at least 70% identity (suitably at least 75%, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to the major U1 snRNA sequence [cloverleaf] (nt 410-562) of the U1_256 sequence described herein. In some embodiments, a modified U1 snRNA of the invention comprises the major U1 snRNA sequence [cloverleaf] (nt 410-562) of the U1_256 sequence described herein. The major U1 snRNA sequence [cloverleaf] (nt 410-562) of the U1_256 sequence (SEQ ID NO: 15) is as follows: [ka]

[0120] In some embodiments, the modified U1 snRNA of the invention comprises the following nucleotide sequence: [ka]

[0121] In some embodiments, the modified U1 snRNA of the invention comprises the following nucleotide sequence: [ka]

[0122] In some embodiments, the modified U1 snRNA of the invention comprises the following nucleotide sequence: [ka]

[0123] In some preferred embodiments, the first 11 nucleotides of the U1 snRNA, which comprise the natural splice donor annealing sequence, may be replaced in whole or in part by a heterologous sequence complementary to a nucleotide sequence within the packaging region of a lentiviral vector genome. Suitably, between 1 and 11 (suitably between 2 and 11, 3 and 11, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) nucleic acids of the first 11 nucleotides of the U1 snRNA are replaced by a heterologous sequence complementary to a nucleotide sequence within the packaging region of a lentiviral vector genome sequence.

[0124] In some embodiments, the native splice donor annealing sequence may be replaced in whole or in part by a heterologous sequence complementary to a nucleotide sequence in the packaging region of the lentiviral vector genome sequence. Suitably, 1 to 11 (suitably 2 to 11, 3 to 11, 5 to 11, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) nucleic acids of the native splice donor annealing sequence are replaced by a heterologous sequence complementary to a nucleotide sequence in the packaging region of the lentiviral vector genome sequence. In a preferred embodiment, the entire native splice donor annealing sequence is replaced by a heterologous sequence complementary to a nucleotide sequence in the packaging region of the lentiviral vector genome sequence. That is, the native splice donor annealing sequence (e.g., 5'-ACUUACCUG-3') is completely replaced by a heterologous sequence in accordance with the present invention.

[0125] In some embodiments, a modified U1 snRNA comprising a heterologous sequence complementary to a nucleotide sequence within the packaging region of a lentiviral vector genome sequence encodes an A at the first nucleotide at the 5' end of the heterologous sequence, regardless of whether the A is involved in annealing to a target sequence.

[0126] In some embodiments, a modified U1 snRNA comprising a heterologous sequence complementary to a nucleotide sequence within the packaging region of a lentiviral vector genome sequence encodes an AU in the first two nucleotides at the 5' end of the heterologous sequence, regardless of whether the A or the U is involved in annealing to the target sequence.

[0127] In some embodiments, a modified U1 snRNA comprising a heterologous sequence complementary to a nucleotide sequence within the packaging region of a lentiviral vector genome sequence does not encode an AU in the first two nucleotides at the 5' end of the heterologous sequence, and the first nucleotide may or may not be involved in annealing to a target sequence.

[0128] In some embodiments, the heterologous sequence complementary to a nucleotide sequence within the packaging region of the lentiviral vector genome sequence comprises at least 7 nucleotides of complementarity to the nucleotide sequence. In some embodiments, the heterologous sequence complementary to a nucleotide sequence within the packaging region of the lentiviral vector genome sequence comprises at least 9 nucleotides of complementarity to the nucleotide sequence. Preferably, the heterologous sequence for use in the present invention comprises 15 nucleotides of complementarity to the nucleotide sequence.

[0129] Suitably, heterologous sequences for use in the present invention may comprise from 7 to 25 (suitably 7 to 20, 7 to 15, 9 to 15, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) nucleotides.

[0130] Suitably, a heterologous sequence for use in the present invention may comprise 7 nucleotides.

[0131] Suitably, a heterologous sequence for use in the present invention may comprise 8 nucleotides.

[0132] Suitably, a heterologous sequence for use in the present invention may comprise 9 nucleotides.

[0133] Suitably, a heterologous sequence for use in the present invention may comprise 10 nucleotides.

[0134] Suitably, a heterologous sequence for use in the present invention may comprise 11 nucleotides.

[0135] Suitably, a heterologous sequence for use in the present invention may comprise 12 nucleotides.

[0136] Suitably, a heterologous sequence for use in the present invention may comprise 13 nucleotides.

[0137] Suitably, a heterologous sequence for use in the present invention may comprise 14 nucleotides.

[0138] Suitably, a heterologous sequence for use in the present invention may comprise 15 nucleotides.

[0139] Suitably, a heterologous sequence for use in the present invention may comprise 16 nucleotides.

[0140] Suitably, a heterologous sequence for use in the present invention may comprise 17 nucleotides.

[0141] Suitably, a heterologous sequence for use in the present invention may comprise 18 nucleotides.

[0142] Suitably, a heterologous sequence for use in the present invention may comprise 19 nucleotides.

[0143] Suitably, a heterologous sequence for use in the present invention may comprise 20 nucleotides.

[0144] Suitably, a heterologous sequence for use in the present invention may comprise 21 nucleotides.

[0145] Suitably, a heterologous sequence for use in the present invention may comprise 22 nucleotides.

[0146] Suitably, a heterologous sequence for use in the present invention may comprise 23 nucleotides.

[0147] Suitably, a heterologous sequence for use in the present invention may comprise 24 nucleotides.

[0148] Suitably, a heterologous sequence for use in the present invention may comprise 25 nucleotides.

[0149] In some embodiments, the nucleotide sequence in the packaging region of the lentiviral vector genome sequence is located within the 5'U5 domain, the PBS element, the SL1 element, the SL2 element, the SL3ψ element, the SL4 element, and / or a sequence derived from the gag gene. Suitably, the nucleotide sequence in the packaging region of the lentiviral vector genome sequence is located within the SL1, SL2, and / or SL3ψ element. In some preferred embodiments, the nucleotide sequence in the packaging region of the lentiviral vector genome sequence is located within the SL1 and / or SL2 element. In some particularly preferred embodiments, the nucleotide sequence in the packaging region of the lentiviral vector genome sequence is located within the SL1 element.

[0150] In some embodiments, the nucleotide sequence within the packaging region of the lentiviral vector genome sequence comprises at least 7 nucleotides. In some embodiments, the nucleotide sequence within the packaging region of the lentiviral vector genome sequence comprises at least 9 nucleotides. Suitably, the nucleotide sequence within the packaging region of the lentiviral vector genome sequence comprises 7 to 25 (suitably 7 to 20, 7 to 15, 9 to 15, 7, 8, 9, 10, 11, 12, 13, 14 or 15) nucleotides.

[0151] Suitably, the nucleotide sequence within the packaging region of the lentiviral vector genome sequence comprises 7 nucleotides.

[0152] Suitably, the nucleotide sequence within the packaging region of the lentiviral vector genome sequence comprises 8 nucleotides.

[0153] Suitably, the nucleotide sequence within the packaging region of the lentiviral vector genome sequence comprises 9 nucleotides.

[0154] Suitably, the nucleotide sequence within the packaging region of the lentiviral vector genome sequence comprises 10 nucleotides.

[0155] Suitably, the nucleotide sequence within the packaging region of the lentiviral vector genome sequence comprises 11 nucleotides.

[0156] Suitably, the nucleotide sequence within the packaging region of the lentiviral vector genome sequence comprises 12 nucleotides.

[0157] Suitably, the nucleotide sequence within the packaging region of the lentiviral vector genome sequence comprises 13 nucleotides.

[0158] Suitably, the nucleotide sequence within the packaging region of the lentiviral vector genome sequence comprises 14 nucleotides.

[0159] Suitably, the nucleotide sequence within the packaging region of the lentiviral vector genome sequence comprises 15 nucleotides.

[0160] Suitably, the nucleotide sequence within the packaging region of the lentiviral vector genome sequence comprises 16 nucleotides.

[0161] Suitably, the nucleotide sequence within the packaging region of the lentiviral vector genome sequence comprises 17 nucleotides.

[0162] Suitably, the nucleotide sequence within the packaging region of the lentiviral vector genome sequence comprises 18 nucleotides.

[0163] Suitably, the nucleotide sequence within the packaging region of the lentiviral vector genome sequence comprises 19 nucleotides.

[0164] Suitably, the nucleotide sequence within the packaging region of the lentiviral vector genome sequence comprises 20 nucleotides.

[0165] Suitably, the nucleotide sequence within the packaging region of the lentiviral vector genome sequence comprises 21 nucleotides.

[0166] Suitably, the nucleotide sequence within the packaging region of the lentiviral vector genome sequence comprises 22 nucleotides.

[0167] Suitably, the nucleotide sequence within the packaging region of the lentiviral vector genome sequence comprises 23 nucleotides.

[0168] Suitably, the nucleotide sequence within the packaging region of the lentiviral vector genome sequence comprises 24 nucleotides.

[0169] Suitably, the nucleotide sequence within the packaging region of the lentiviral vector genome sequence comprises 25 nucleotides.

[0170] Preferably, the nucleotide sequence within the packaging region of the lentiviral vector genome sequence comprises 15 nucleotides.

[0171] Binding of the modified U1 snRNA of the present invention to a nucleotide sequence within the packaging region of a lentiviral vector genome sequence can increase lentiviral vector titer during lentiviral vector production compared to lentiviral vector production in the absence of the modified U1 snRNA of the present invention. Thus, production of a lentiviral vector in the presence of the modified U1 snRNA of the present invention increases lentiviral vector titer compared to lentiviral vector production in the absence of the modified U1 snRNA of the present invention. Suitable assays for measuring lentiviral vector titer are described herein. Suitably, lentiviral vector production involves coexpression of the modified U1 snRNA with vector components including the gag, env, rev, and RNA genome of the lentiviral vector. In some embodiments, the increase in lentiviral vector titer occurs in the presence or absence of a functional 5' LTR polyA site. In some embodiments, the increase in lentiviral vector titer conferred by the modified U1 snRNA of the present invention is independent of polyA site suppression in the 5' LTR of the vector genome.

[0172] In some embodiments, binding of the modified U1 snRNA of the present invention to a nucleotide sequence within the packaging region of a lentiviral vector genome sequence may increase lentiviral vector titer during lentiviral vector production by at least 30% compared to lentiviral vector production in the absence of the modified U1 snRNA of the present invention. Suitably, binding of the modified U1 snRNA of the present invention to a nucleotide sequence within the packaging region of a lentiviral vector genome sequence may increase lentiviral vector titer during lentiviral vector production by at least 35% (suitably by at least 40%, 45%, 50%, 60%, 70%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950% or 1000%) compared to lentiviral vector production in the absence of the modified U1 snRNA of the present invention.

[0173] The modified U1 snRNA of the present invention can be designed by (a) selecting a target site (a preselected nucleotide site) in the packaging region of the lentiviral vector genome for binding to the modified U1 snRNA, and (b) introducing a heterologous sequence within the natural splice donor annealing sequence at the 5' end of the U1 snRNA (e.g., 5'-ACUUACCUG-3') that is complementary to the preselected nucleotide site selected in step (a).

[0174] It is within the ability of one of ordinary skill in the art to introduce a heterologous sequence complementary to a target site into or in place of the natural splice donor annealing sequence (e.g., 5'-ACUUACCUG-3') at the 5' end of the endogenous U1 snRNA using conventional techniques of molecular biology. Generally speaking, suitable conventional methods include directed mutagenesis or substitution by homologous recombination.

[0175] It is within the ability of one of ordinary skill in the art to modify the natural splice donor annealing sequence at the 5' end of the endogenous U1 snRNA (e.g., 5'-ACUUACCUG-3') so that it has the same sequence as the heterologous sequence complementary to the target site, using standard techniques of molecular biology. For example, suitable methods include directed or random mutagenesis, followed by selection for mutations that result in modified U1 snRNAs according to the present invention.

[0176] The modified U1 snRNA of the present invention can be produced according to methods generally known in the art, for example, the modified U1 snRNA can be produced by chemical synthesis or recombinant DNA / RNA technology.

[0177] It is within the capabilities of one of ordinary skill in the art to introduce into cells a nucleotide sequence encoding the modified U1 snRNA of the present invention using conventional molecular and cell biology techniques. For example, an expression cassette can be used, as described below.

[0178] Thus, in another aspect, the present invention provides a cell comprising the modified U1 snRNA of the present invention. Suitable cells are described below.

[0179] In another aspect, as demonstrated in the Examples herein, the modified U1 according to the invention described herein can beneficially provide for the repression of transgene expression. Accordingly, the present invention also includes a method for transgene repression using modified U1 as described herein, or a method or use thereof.

[0180] Nucleotide sequence In another aspect, the present invention provides a nucleotide sequence encoding a modified U1 snRNA of the present invention.

[0181] The term "nucleotide sequence" in the context of the present invention can be a double-stranded or single-stranded molecule and includes genomic DNA, cDNA, synthetic DNA, RNA, and chimeric DNA / RNA molecules. Preferably, it refers to a DNA, more preferably a cDNA, sequence encoding the modified U1 snRNA of the present invention.

[0182] Typically, nucleotide sequences within the scope of the present invention are produced using recombinant DNA techniques (ie, recombinant DNA) as described herein.

[0183] In a preferred embodiment, the nucleotide sequence encoding the modified U1 snRNA of the present invention is an expression cassette.

[0184] The presence / abundance of modified U1 snRNA molecules can be quantified in vector-producing cell extracts or vector virions by extraction of total RNA followed by RT-PCR or RT-qPCR (quantitative) using DNA primers. Importantly, the forward primer is designed to have complementarity to the targeted sequence of the modified U1 snRNA molecule, so that only the modified U1 snRNA, and not the endogenous U1 snRNA, is amplified during qPCR.

[0185] In one aspect, the present invention provides a vector virion comprising a modified U1 according to the invention as described herein.

[0186] Vectors / Expression Cassettes A vector is a means that allows or facilitates the transfer of an entity from one environment to another. In the present invention, for example, some vectors used in recombinant nucleic acid technology allow an entity, such as a segment of nucleic acid (e.g., a heterologous DNA segment, e.g., a heterologous cDNA segment), to be introduced into and expressed by a target cell. The vector can facilitate the integration of a nucleotide sequence encoding a modified U1 snRNA (or viral vector component) of the present invention and maintain the nucleotide sequence encoding the modified U1 snRNA (or viral vector component) of the present invention and its expression in the target cell.

[0187] A vector can be or contain an expression cassette (also called an expression construct). An expression cassette as described herein comprises a region of nucleic acid that contains a sequence that can be transcribed. Thus, sequences encoding mRNA, tRNA, and rRNA are included in this definition.

[0188] Vectors may contain one or more selectable marker genes (e.g., a neomycin resistance gene) and / or traceable marker genes (e.g., a gene encoding green fluorescent protein (GFP)). Vectors may be used, for example, to infect and / or transduce target cells. Vectors may further contain nucleotide sequences that enable the vector to replicate in the host cell of interest.

[0189] The term "cassette" is synonymous with terms such as "conjugate," "construct," and "hybrid," and comprises a polynucleotide sequence linked directly or indirectly to a promoter. Expression cassettes of the present invention comprise a promoter for expression of a nucleotide sequence encoding a modified U1 snRNA of the present invention, and may optionally comprise a regulator of the nucleotide sequence encoding the modified U1 snRNA of the present invention. Expression cassettes for use in the present invention comprise a promoter for expression of a nucleotide sequence encoding a viral vector component, and may optionally comprise a regulator of the nucleotide sequence encoding the viral vector component. Preferably, the cassette comprises at least a polynucleotide sequence operably linked to a promoter.

[0190] The expression cassette can be used to replicate a nucleotide sequence encoding the modified U1 snRNA of the present invention in a compatible target cell in vitro. Thus, the present invention provides a method for producing modified U1 snRNA in vitro by introducing an expression cassette of the present invention into a compatible target cell in vitro and growing the target cell under conditions that result in expression of the modified U1 snRNA. The modified U1 snRNA can be recovered from the target cell by methods well known in the art. Suitable target cells include mammalian cell lines and other eukaryotic cell lines.

[0191] The choice of expression cassette, e.g., a plasmid, cosmid, virus, or phage vector, often depends on the host cell into which it will be introduced. The expression cassette can be a DNA plasmid (supercoiled, nicked, or linear), minicircle DNA (linear or supercoiled), plasmid DNA containing only the region of interest by removal of the plasmid backbone by restriction enzyme digestion and purification, or DNA generated using an enzymatic DNA amplification platform such as doggybone DNA (dbDNA™) (in which case the final DNA used is in a closed ligated form, or it has been prepared to have open cut ends (e.g., by restriction enzyme digestion)).

[0192] Thus, in one aspect, the present invention provides an expression cassette comprising a nucleotide sequence encoding a modified U1 snRNA of the present invention.

[0193] In one embodiment of the present invention, the modified U1 expression cassette described herein can be delivered to the cells described herein by a lentiviral or retroviral vector.

[0194] It is within the capabilities of one skilled in the art to introduce the expression cassette of the present invention into cells using conventional molecular and cell biology techniques.

[0195] In another aspect, the present invention provides a cell comprising an expression cassette of the present invention. Suitable cells are described below.

[0196] Lentiviral vector production system and cells A lentiviral vector production system comprises a set of nucleotide sequences encoding the components necessary for the production of a lentiviral vector. Thus, a vector production system comprises a set of nucleotide sequences encoding the viral vector components necessary for the generation of lentiviral vector particles.

[0197] A "viral vector production system" or "vector production system" or "production system" should be understood as a system that contains the components necessary for lentiviral vector production.

[0198] In one embodiment, the nucleotide sequence may be suitable for use in a lentiviral vector in a tat-independent system for vector production. As described herein, third-generation lentiviral vectors are U3-dependent (and use a heterologous promoter to drive transcription), and the nucleotide sequence according to the present invention may be used in the context of a third-generation lentiviral vector. In one embodiment of the present invention, tat is not provided in the lentiviral vector production system, e.g., tat is not provided in trans. In one embodiment, the cells or vectors or vector production systems described herein do not contain tat protein.

[0199] In one aspect, the present invention provides a nucleotide sequence encoding the RNA genome of a lentiviral vector, wherein a major splice donor site in the RNA genome of the lentiviral vector is inactivated and a cryptic splice donor 3' to the major splice donor site is inactivated, and the nucleotide sequence is for use in a Tat-independent lentiviral vector.

[0200] In one aspect, the present invention provides a nucleotide sequence encoding the RNA genome of a lentiviral vector, wherein a major splice donor site in the RNA genome of the lentiviral vector is inactivated and a cryptic splice donor site 3' to the major splice donor site is inactivated, and wherein the nucleotide sequence is produced in the absence of tat.

[0201] In one aspect, the present invention provides a nucleotide sequence encoding the RNA genome of a lentiviral vector, wherein a major splice donor site in the RNA genome of the lentiviral vector is inactivated, a cryptic splice donor site 3' to the major splice donor site is inactivated, and the nucleotide sequence is transcribed in a tat-independent manner.

[0202] In one aspect, the present invention provides a nucleotide sequence encoding the RNA genome of a lentiviral vector, wherein a major splice donor site in the RNA genome of the lentiviral vector is inactivated and a cryptic splice donor site 3' to the major splice donor site is inactivated, and the nucleotide sequence is for use in a U3-independent lentiviral vector.

[0203] In one aspect, the present invention provides a nucleotide sequence encoding the RNA genome of a lentiviral vector, wherein a major splice donor site in the RNA genome of the lentiviral vector is inactivated and a cryptic splice donor site 3' to the major splice donor site is inactivated, and the nucleotide sequence is transcribed independently of a U3 promoter.

[0204] In one aspect, the present invention provides a nucleotide sequence encoding the RNA genome of a lentiviral vector, wherein a major splice donor site in the RNA genome of the lentiviral vector is inactivated and a cryptic splice donor site 3' to the major splice donor site is inactivated, and the nucleotide sequence is transcribed by a heterologous promoter.

[0205] In one embodiment, transcription of the nucleotide sequences described herein is not dependent on the presence of U3. The nucleotide sequences may be derived from a U3-independent transcription event. The nucleotide sequences may be derived from a heterologous promoter. The nucleotide sequences described herein may not contain a native U3 promoter.

[0206] In one embodiment, the viral vector production system comprises nucleotide sequences encoding the Gag and Gag / Pol proteins, as well as the Env protein and vector genome sequence. The production system may optionally include a nucleotide sequence encoding the Rev protein or a functional replacement thereof.

[0207] In one embodiment of the invention, at least one transgene component may be inverted or in the reverse orientation.

[0208] In one embodiment, at least one transgene component can be inverted or reverse orientation relative to the 5'-3' orientation of the vector genome RNA. Lentiviral vector genomes can be used in which the transgene cassette is inverted, i.e., the transcription unit is opposite to the promoter driving the vector genome cassette. It is also possible for a component of the transgene cassette to be in the reverse orientation and another component to be in the forward orientation, for example, allowing the use of a bidirectional transgene cassette or multiple separate cassettes.

[0209] In one embodiment, the viral vector production system includes a modular nucleic acid construct (modular construct). A modular construct is a DNA expression construct containing two or more nucleic acids used in the production of lentiviral vectors. A modular construct can be a DNA plasmid containing two or more nucleic acids used in the production of lentiviral vectors. The plasmid can be a bacterial plasmid. The nucleic acid can encode, for example, gag-pol, rev, env, or a vector genome. Modular constructs designed for the generation of packaging and producer cell lines should also encode transcriptional regulatory proteins (e.g., TetR, CymR) and / or translational repressor proteins (e.g., TRAP) and selectable markers (e.g., Zeocin™, hygromycin, blasticidin, puromycin, or neomycin resistance genes). Suitable modular constructs for use in the present invention are described in EP 3502260, which is incorporated herein by reference in its entirety.

[0210] Because modular constructs for use according to the present invention contain nucleic acid sequences encoding two or more of the retroviral components on a single construct, the safety profile of these modular constructs has been considered, and additional safety features have been incorporated directly into the constructs. These features include the use of insulators for the multiple open reading frames of the retroviral vector components and / or the specific orientation and placement of the retroviral genes in the modular construct. It is believed that the use of these features prevents direct read-through to generate replication-competent viral particles.

[0211] Nucleic acid sequences encoding viral vector components may be in reverse and / or alternate transcriptional orientation in a modular construct. Thus, the nucleic acid sequences encoding viral vector components are not provided in the same 5' to 3' orientation, and as a result, viral vector components cannot be generated from the same mRNA molecule. Reverse orientation may mean that at least two coding sequences for different vector components are provided in a "head-to-head" and "tail-to-tail" transcriptional orientation. This may be achieved by providing the coding sequence of one vector component (e.g., env) on one strand of the modular construct and the coding sequence of another vector component (e.g., rev) on the opposite strand. Preferably, when coding sequences for three or more vector components are present in a modular construct, at least two of the coding sequences are present in reverse transcriptional orientation. Thus, when coding sequences for three or more vector components are present in a modular construct, each component may be oriented so that it is present in the opposite 5' to 3' orientation relative to all of the adjacent coding sequences of the other vector components to which it is adjacent. That is, alternate 5' to 3' (or transcriptional) orientations for each coding sequence may be used.

[0212] Modular constructs for use according to the invention may comprise nucleic acid sequences encoding two or more of the following vector components: gag-pol, rev, env, vector genome. Modular constructs may comprise nucleic acid sequences encoding any combination of vector components. In one embodiment, modular constructs may comprise nucleic acid sequences encoding: i) the RNA genome and rev of the retroviral vector, or their functional substitutes; ii) the RNA genome and gag-pol of the retroviral vector; iii) the RNA genome and env of the retroviral vector; iv) gag-pol and rev, or their functional substitutes; v) gag-pol and env; vi) env and rev, or their functional substitutes; vii) the RNA genome of the retroviral vector, rev or their functional substitutes, and gag-pol; viii) the RNA genome of the retroviral vector, rev, or a functional substitute thereof, and env; ix) the RNA genome, gag-pol and env of a retroviral vector; or x) gag-pol, rev, or their functional replacements, and env (wherein the nucleic acid sequences are in reverse and / or alternate orientation).

[0213] In one embodiment, a cell for producing a retroviral vector can contain nucleic acid sequences encoding any of the combinations of i) to x) above, where the nucleic acid sequences are located at the same locus and in reverse and / or alternate orientation. The same locus can refer to a single extrachromosomal locus within the cell, such as a single plasmid, or a single locus within the genome of the cell (i.e., a single insertion site). The cell can be a stable or transient cell for producing a retroviral vector, such as a lentiviral vector.

[0214] DNA expression constructs can be DNA plasmids (supercoiled, nicked, or linear), minicircle DNA (linear or supercoiled), plasmid DNA containing only the region of interest by removal of the plasmid backbone by restriction enzyme digestion and purification, DNA generated using enzymatic DNA amplification platforms such as doggybone DNA (dbDNA™) (in this case, the final DNA used is in a closed ligated form, or it has been prepared to have open cut ends (e.g., by restriction enzyme digestion)).

[0215] In one embodiment, the lentiviral vector is derived from an HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or visna lentivirus.

[0216] "Viral vector producer cells," "vector producer cells," or "producer cells" should be understood as cells capable of producing lentiviral vectors or lentiviral vector particles. Lentiviral vector producer cells can be "producer cells" or "packaging cells." One or more DNA constructs of the viral vector system can be stably integrated into the viral vector producer cells or can be episomally maintained in the producer cells. Alternatively, all of the DNA components of the viral vector system can be transiently transfected into the viral vector producer cells. In yet another alternative embodiment, producer cells stably expressing some of the components can be transiently transfected with the remaining components required for vector production.

[0217] In one embodiment of the invention described herein, the U1 expression cassette is stably integrated into the cells of the invention described herein.

[0218] As used herein, the term "packaging cell" refers to a cell that contains the elements necessary for the production of lentiviral vector particles but lacks a vector genome. Optionally, such packaging cells may contain one or more expression cassettes capable of expressing viral structural proteins (e.g., gag, gag / pol, and env) and typically rev.

[0219] The producer / packaging cells can be of any suitable cell type. Producer cells are generally mammalian cells, but can also be, for example, insect cells.

[0220] As used herein, the term "producer cell" or "vector production / producer cell" refers to a cell that contains all of the elements necessary for the production of a lentiviral vector particle. Producer cells can be stable producer cell lines or transiently derived, or can be stable packaging cells in which the retroviral genome is transiently expressed.

[0221] Vector-producing cells can be cells cultured in vitro, such as tissue culture cell lines. Suitable cell lines include, but are not limited to, mammalian cells, such as mouse fibroblast-derived cell lines or human cell lines. Preferably, vector-producing cells are derived from human cell lines.

[0222] Cells and production methods Another aspect of the present invention relates to a method for producing a lentiviral vector, comprising introducing a nucleotide sequence described herein into a cell (e.g., a producer cell) and culturing the cell under conditions suitable for the production of the lentiviral vector.

[0223] Thus, in one aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis. a) introducing into a cell nucleotide sequences encoding the vector components and at least one nucleotide sequence encoding a modified U1 snRNA of the invention; b) optionally, selecting cells containing said nucleotide sequences encoding the vector components and at least one nucleotide sequence encoding a modified U1 snRNA of the invention; c) further culturing the cells under conditions whereby the vector components are co-expressed with the modified U1 snRNA and a lentiviral vector is produced; and d) optionally isolating the lentiviral vector. The present invention provides a method for producing a lentiviral vector, comprising:

[0224] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) introducing into a cell nucleotide sequences encoding the vector components and at least one nucleotide sequence encoding a modified U1 snRNA of the invention; b) selecting cells containing the nucleotide sequences encoding the vector components and at least one nucleotide sequence encoding a modified U1 snRNA of the invention; c) optionally introducing into said selected cells a nucleic acid vector different from the nucleotide sequence encoding the modified U1 snRNA of the present invention; d) further culturing the cells under conditions in which the lentiviral vector is produced; and e) optionally isolating the lentiviral vector. The present invention provides a method for producing a lentiviral vector, comprising:

[0225] In the methods of the present invention, the vector components may include gag, env, rev, and / or the RNA genome of a lentiviral vector. The nucleotide sequences encoding the vector components and at least one nucleotide sequence encoding a modified U1 snRNA of the present invention may be introduced into a cell simultaneously or sequentially in any order. The nucleotide sequences encoding the vector components may be introduced into a cell before the at least one nucleotide sequence encoding a modified U1 snRNA of the present invention. The at least one nucleotide sequence encoding a modified U1 snRNA of the present invention may be introduced into a cell before the nucleotide sequences encoding the vector components.

[0226] In some embodiments, the lentiviral vector can be replication-defective.

[0227] In another aspect, the invention provides a lentiviral vector produced by any of the methods of the invention.

[0228] In another aspect, the present invention provides the use of a modified U1 snRNA of the invention or a nucleotide sequence encoding the modified U1 snRNA of the invention or a producer cell of the invention for the manufacture of a lentiviral vector.

[0229] The production of lentiviral vectors may involve co-expression of the modified U1 snRNA of the invention with the vector components in suitable producer cells as described herein.

[0230] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) introducing into a cell nucleotide sequences encoding vector components including gag, env, rev, and the RNA genome of a lentiviral vector, and at least one nucleotide sequence encoding a modified U1 snRNA of the present invention; and b) optionally, selecting cells containing said nucleotide sequences encoding the vector components and at least one nucleotide sequence encoding a modified U1 snRNA of the invention. The present invention provides a method for producing a producer cell for producing a lentiviral vector, comprising:

[0231] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) introducing into a cell nucleotide sequences encoding vector components including gag, env, rev, and the RNA genome of a lentiviral vector, and at least one nucleotide sequence encoding a modified U1 snRNA of the present invention; and b) selecting cells containing said nucleotide sequences encoding the vector components or at least one nucleotide sequence encoding at least one modified U1 snRNA of the invention; The present invention provides a method for producing a stable producer cell for producing a lentiviral vector, comprising:

[0232] In another aspect, the present invention provides a method for producing a transient production cell for producing a lentiviral vector, comprising introducing into a cell a nucleotide sequence encoding vector components including gag, env, rev and the RNA genome of a lentiviral vector, and at least one nucleotide sequence encoding a modified U1 snRNA of the present invention.

[0233] In another aspect, the present invention provides a cell for producing a lentiviral vector produced by any of the methods of the present invention.

[0234] In another aspect, the present invention provides a stable producer cell for producing a lentiviral vector produced by any of the methods of the present invention.

[0235] In another aspect, the present invention provides a transient production cell for producing a lentiviral vector produced by any of the methods of the present invention.

[0236] In another aspect, the present invention provides a cell for producing a lentiviral vector comprising at least one nucleotide sequence encoding a modified U1 snRNA of the present invention.

[0237] In another aspect, the present invention provides a stable producer cell for producing a lentiviral vector comprising at least one nucleotide sequence encoding a modified U1 snRNA of the present invention.

[0238] In another aspect, the present invention provides a transient production cell for producing a lentiviral vector comprising at least one nucleotide sequence encoding a modified U1 snRNA of the present invention.

[0239] In some embodiments, stable or transient producer cells for producing lentiviral vectors contain 1, 5, 10, 15, 20, or 30 stably integrated nucleotide sequences encoding the modified U1 snRNA of the invention.

[0240] In some embodiments of the methods and uses of the present invention, binding of the modified U1 snRNA of the present invention to a nucleotide sequence within the packaging region of a lentiviral vector genome sequence increases lentiviral vector titer during lentiviral vector production compared to lentiviral vector production in the absence of the modified U1 snRNA of the present invention. Accordingly, production of a lentiviral vector in the presence of the modified U1 snRNA of the present invention increases lentiviral vector titer compared to lentiviral vector production in the absence of the modified U1 snRNA of the present invention. Suitable assays for measuring lentiviral vector titer are described herein. Suitably, lentiviral vector production involves coexpression of the modified U1 snRNA with vector components including gag, env, rev, and the lentiviral vector RNA genome. In some embodiments, the increase in lentiviral vector titer occurs in the presence or absence of a functional 5' LTR polyA site. In some embodiments, the increase in lentiviral vector titer conferred by the modified U1 snRNA of the present invention is independent of polyA site suppression in the 5' LTR of the vector genome.

[0241] In some embodiments of the methods and uses of the present invention, binding of the modified U1 snRNA of the present invention to a nucleotide sequence within the packaging region of a lentiviral vector genome sequence may increase lentiviral vector titer during lentiviral vector production by at least 30% compared to lentiviral vector production in the absence of the modified U1 snRNA of the present invention. Suitably, binding of the modified U1 snRNA of the present invention to a nucleotide sequence within the packaging region of a lentiviral vector genome sequence may increase lentiviral vector titer during lentiviral vector production by at least 35% (suitably by at least 40%, 45%, 50%, 60%, 70%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950% or 1000%) compared to lentiviral vector production in the absence of the modified U1 snRNA of the present invention.

[0242] In some embodiments of the methods and uses of the present invention, suitable producer cells or cells for producing lentiviral vectors are cells capable of producing viral vectors or viral vector particles when cultured under appropriate conditions. Thus, the cells typically contain nucleotide sequences encoding vector components, which may include gag, env, rev, and the RNA genome of the lentiviral vector. Suitable cell lines include, but are not limited to, mammalian cells, such as mouse fibroblast-derived cell lines or human cell lines. These are generally mammalian cells, including human cells, such as HEK293T, HEK293, CAP, CAP-T, or CHO cells, but can also be insect cells, such as SF9 cells. Preferably, vector-producing cells are derived from a human cell line. Therefore, such suitable producer cells can be used in any of the methods or uses of the present invention.

[0243] Methods for introducing nucleotide sequences into cells are well known in the art and have been described. Thus, it is within the capabilities of a person skilled in the art to introduce nucleotide sequences encoding vector components, including gag, env, rev, and the RNA genome of a lentiviral vector, and at least one nucleotide sequence encoding the modified U1 snRNA of the present invention or at least one expression cassette of the present invention into cells using conventional techniques in molecular and cell biology.

[0244] Stable production cells can be packaging or producer cells. To generate producer cells from packaging cells, vector genome DNA constructs can be stably or transiently introduced. Packaging / producer cells can be generated by transducing an appropriate cell line with a retroviral vector expressing one of the vector components, i.e., the genome, gag-pol components, and envelope, as described in WO 2004 / 022761.

[0245] Alternatively, nucleotide sequences can be transfected into cells, followed by rare, random integration into the producer cell genome. Transfection methods can be performed using methods well known in the art. For example, stable transfection methods can use constructs engineered to facilitate concatemerization. In another example, transfection can be performed using calcium phosphate or commercially available preparations such as Lipofectamine™ 2000CD (Invitrogen, CA), FuGENE® HD, or polyethyleneimine (PEI). Alternatively, nucleotide sequences can be introduced into producer cells by electroporation. Those skilled in the art will recognize methods for facilitating integration of nucleotide sequences into producer cells. For example, linearization of the nucleic acid construct can be useful if it is naturally circular. Less random integration methods can involve nucleic acid constructs containing regions of homology shared with the endogenous chromosome of the mammalian host cell to direct integration into a selected site within the endogenous genome. Furthermore, if recombination sites are present on the construct, they can be used for targeted recombination. For example, the nucleic acid construct can contain loxP sites, which allow targeted integration when combined with Cre recombinase (i.e., using the Cre / lox system from P1 bacteriophage). Alternatively or additionally, the recombination sites can be att sites (e.g., from λ phage), in which case the att sites allow site-specific integration in the presence of lambda integrase. This allows the lentiviral genes to be targeted to loci within the host cell genome, which allows for high and / or stable expression.

[0246] Other methods of targeted integration are well known in the art. For example, methods that induce targeted cleavage of genomic DNA can be used to promote targeted recombination at a selected chromosomal locus. These methods often involve the use of methods or systems that induce double-strand breaks (DSBs), such as nicks, in the endogenous genome to induce repair of the break by physiological mechanisms such as non-homologous end joining (NHEJ). Cleavage can occur through the use of specific nucleases, such as engineered zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), the CRISPR / Cas9 system in the presence of engineered crRNA / tracr RNA ("single guide RNA") to direct the specific cleavage, and / or nucleases based on the Argonaute system (e.g., from T. thermophilus).

[0247] Packaging / producer cell lines can be generated by integration of nucleotide sequences using lentiviral transduction alone or nucleic acid transfection alone or a combination of both methods.

[0248] Methods for producing retroviral vectors from producer cells, in particular the processing of retroviral vectors, are described in WO2009 / 153563.

[0249] In one embodiment, the production cell may contain an RNA binding protein (eg, tryptophan RNA-binding attenuation protein; TRAP) and / or a Tet repressor (TetR) protein or an alternative regulatory protein (eg, CymR).

[0250] Production of lentiviral vectors from producer cells can be by transfection methods, production from stable cell lines which may include an induction step (e.g., doxycycline induction), or a combination of both. Transfection methods can be performed using methods well known in the art, examples of which have been described above.

[0251] The production cells (either packaging or producer cell lines, or those transiently transfected with lentiviral vectors encoding the components) are cultured to increase the number of cells and virus and / or viral titer. The cells are cultured to allow them to metabolize, grow, divide, and / or produce the desired viral vector of the present invention. This can be achieved by methods well known to those of skill in the art, including, but not limited to, providing nutrients to the cells in an appropriate medium. The method may include growth attached to a surface, growth in suspension, or a combination thereof. Culturing can be performed, for example, in tissue culture flasks, tissue culture multi-well plates, dishes, roller bottles, wave bags, or bioreactors, using batch, fed-batch, continuous, etc. To achieve large-scale production of viral vectors by cell culture, it is preferred in the art that the cells be able to grow in suspension. Suitable conditions for culturing cells are known (see, for example, Tissue Culture, Academic Press, Kruse and Paterson, eds. (1973), and R.I. Freshney, Culture of animal cells: A manual of basic technique, fourth edition (Wiley-Liss Inc., 2000, ISBN 0-471-34889-9)).

[0252] Preferably, the cells are first "bulked up" in tissue culture flasks or bioreactors and then expanded in multi-layer culture vessels or large bioreactors (greater than 50 L) to obtain vector-producing cells of the invention.

[0253] Preferably, the cells are grown in an adherent manner to obtain cells producing the vectors of the present invention.

[0254] Preferably, the cells are grown in suspension to obtain the vector producing cells of the invention.

[0255] Major splice donor Mutations of the major splice donor site in the packaging region of the RNA genome of viral vectors have been shown to be detrimental to vector production titers and, in addition, activate the cryptic splice donor (crSD) immediately adjacent to the MSD. Aberrant splicing from the MSD or CrSD leads to the generation of spliced ​​RNA that cannot be packaged into vector virions. Splicing from the MSD into cellular transcripts from transcriptional readthrough products derived from integrated vectors in transduced cells has also been reported, raising safety concerns. The inventors describe novel mutations within the MSD splicing region that result in a less pronounced decrease in vector titer (in the absence of modified U1 snRNA), leading to a further increase in titer in the presence of modified U1 snRNA. Such mutations or deletions of the major splice donor site may result in additional improvements in vector titer beyond those described herein and may be used in combination with any other aspect of the invention described herein.

[0256] RNA splicing is catalyzed by a large RNA-protein complex called the spliceosome, which is composed of five small nuclear ribonucleoproteins (snRNPs). The boundaries between introns and exons are marked by specific nucleotide sequences within the pre-mRNA, which indicate where splicing occurs. Such boundaries are called "splice sites." The term "splice site" refers to a polynucleotide that can be recognized by the splicing machinery of a eukaryotic cell as suitable for cleavage and / or joining to an alternative splice site.

[0257] Splice sites allow for the excision of introns present in pre-mRNA transcripts. Typically, the 5' splice boundary is referred to as a "splice donor site" or "5' splice site," and the 3' splice boundary is referred to as a "splice acceptor site" or "3' splice site." Splice sites include, for example, naturally occurring splice sites, engineered splice sites, or synthetic splice sites, canonical or consensus splice sites, and / or non-canonical splice sites, such as cryptic splice sites.

[0258] A splice acceptor site generally consists of three distinct sequence elements: a branch point or branch site, a polypyrimidine tract, and an acceptor consensus sequence. The branch point consensus sequence in eukaryotes is YNYTRAC (where Y is a pyrimidine, N is any nucleotide, and R is a purine). The 3' acceptor splice site consensus sequence is YAG (where Y is a pyrimidine) (see, for example, Griffiths et al., eds., Modern Genetic Analysis, 2nd edition, W.H. Freeman and Company, New York (2002)). The 3' splice acceptor site is typically located at the 3' end of an intron.

[0259] Thus, the major splice donor site may be inactivated in the nucleotide sequence encoding the RNA genome of a lentiviral vector for use in the present invention.

[0260] In one aspect, the present invention also provides a cell according to the invention described herein, wherein the major splice donor site in the RNA genome of the lentiviral vector is inactivated, e.g., mutated or deleted.

[0261] In one embodiment, a nucleotide sequence encoding the RNA genome of a lentiviral vector is provided, wherein a major splice donor site in the RNA genome of the lentiviral vector is inactivated, eg, mutated or deleted.

[0262] The terms "canonical splice site" or "consensus splice site" can be used interchangeably and can refer to a splice site that is conserved among species.

[0263] Consensus sequences for the 5' donor splice site and 3' acceptor splice site used in eukaryotic RNA splicing are well known in the art. These consensus sequences contain the nearly invariant dinucleotides at each end of the intron: GT at the 5' end of the intron and AG at the 3' end of the intron.

[0264] A canonical splice donor site consensus sequence (in the case of DNA) can be AG / GTRAGT (where A is adenosine, T is thymine, G is guanine, C is cytosine, R is purine, and " / " indicates the cleavage site). This conforms to the more common splice donor consensus sequence MAGGURR described herein. It is well known in the art that splice donor sequences can deviate from this consensus, particularly in viral genomes where other constraints, such as secondary structure within the vRNA packaging region, affect that same sequence. Non-canonical splice sites are also well known in the art, but are rarer than canonical splice donor consensus sequences.

[0265] "Major splice donor site" refers to the first (predominant) splice donor site in the viral vector genome, which is typically encoded and integrated within the native viral RNA packaging sequence located in the 5' region of the viral vector nucleotide sequence.

[0266] In one embodiment, the nucleotide sequence does not contain an active major splice donor site, i.e., splicing does not occur from the major splice donor site in the nucleotide sequence and splicing activity from the major splice donor site is eliminated.

[0267] The major splice donor site is located in the 5' packaging region of the lentiviral genome.

[0268] In the case of the HIV-1 virus, the major splice donor consensus sequence (in the case of DNA) is TG / GTRAGT (where A is adenosine, T is thymine, G is guanine, C is cytosine, R is purine, and " / " indicates the cleavage site).

[0269] In one embodiment of the invention, the splice donor region, i.e., the region of the vector genome that contains the major splice donor site before the mutation, may have the following sequence: GGGGCGGCGACTGGTGAGTACGCCAAAAAT (SEQ ID NO: 1).

[0270] In one embodiment of the invention, the mutated splice donor region may comprise the following sequence: GGGGCGGCGACTGCAGACAACGCCAAAAAT (SEQ ID NO: 2 - MSD-2KO).

[0271] In one embodiment of the invention, the mutated splice donor region may comprise the following sequence: GGGGCGGCGAGTGGAGACTACGCCAAAAAT (SEQ ID NO: 11 - MSD-2KOv2).

[0272] In one embodiment of the invention, the mutated splice donor region may comprise the following sequence: GGGGAAGGCAACAGATAAATATGCCTTAAAAT (SEQ ID NO: 12 - MSD-2KOm5).

[0273] In one embodiment of the invention, prior to modification, the splice donor region may comprise the following sequence: GGCGACTGGTGAGTACGCC (SEQ ID NO: 9).

[0274] This sequence is also referred to herein as the "stem-loop 2" region (SL2). This sequence can form a stem-loop structure in the splice donor region of the vector genome. In one embodiment of the present invention, this sequence (SL2) can be deleted from the nucleotide sequence according to the present invention described herein.

[0275] Thus, the present invention includes nucleotide sequences that do not include SL2. The present invention includes nucleotide sequences that do not include the sequence of SEQ ID NO:9.

[0276] In one embodiment of the invention, the major splice donor site has the following consensus sequence: TG / GTRAGT (SEQ ID NO: 3) (where R is a purine and " / " is a cleavage site).

[0277] In one embodiment, R can be guanine (G).

[0278] In one embodiment of the invention, the major splice donor and cryptic splice donor regions have the following core sequence: / GTGA / GTA (SEQ ID NO: 13) (where " / " is the site of cleavage at the major splice donor and cryptic splice donor sites).

[0279] In one embodiment of the present invention, the MSD mutant vector genome can have at least two mutations in the major splice donor and cryptic splice donor "regions" (SEQ ID NO: 13), where the first and second "GT" nucleotides are located immediately 3' to the major splice donor and cryptic splice donor nucleotides, respectively.

[0280] In one embodiment of the invention, the major splice donor consensus sequence is CTGGT (SEQ ID NO: 4). The major splice donor site may contain the sequence CTGGT.

[0281] In one embodiment, the nucleotide sequence prior to splice site inactivation comprises the sequence set forth in any of SEQ ID NOs: 1, 3, 4, 9, 10 and / or 13.

[0282] In one embodiment, the nucleotide sequence comprises an inactivated major splice donor site which, when not inactivated, has a cleavage site between nucleotides corresponding to nucleotides 13 and 14 of SEQ ID NO:1.

[0283] In the invention described herein, the nucleotide sequence also contains an inactive cryptic splice donor site. In one embodiment, the nucleotide sequence does not contain an active cryptic splice donor site adjacent to (3' to) the major splice donor site, i.e., no splicing occurs from the adjacent cryptic splice donor site, and splicing from the cryptic splice donor site is eliminated.

[0284] The term "cryptic splice donor site" refers to a nucleic acid sequence that normally does not function as a splice donor site or is less efficiently utilized as a splice donor site due to its adjacent sequence context (e.g., the presence of a nearby "preferred" splice donor), but can be activated to become a more efficiently functioning splice donor site by mutation of the adjacent sequence (e.g., mutation of the nearby "preferred" splice donor).

[0285] In one embodiment, the cryptic splice donor site is the first cryptic splice donor site 3' to the major splice donor.

[0286] In one embodiment, the cryptic splice donor site is located within 6 nucleotides of the major splice donor site 3' to the major splice donor site. Preferably, the cryptic splice donor site is located within 4 or 5 nucleotides, preferably within 4 nucleotides, of the major splice donor cleavage site.

[0287] In one embodiment of the invention, the cryptic splice donor site has the consensus sequence TGAGT (SEQ ID NO: 10).

[0288] In one embodiment, the nucleotide sequence comprises an inactivated cryptic splice donor site which, when not inactivated, has a cleavage site between nucleotides corresponding to nucleotides 17 and 18 of SEQ ID NO:1.

[0289] In one embodiment of the invention, the major splice donor site and / or the adjacent cryptic splice donor site contain a "GT" motif. In one embodiment of the invention, both the major splice donor site and the adjacent cryptic splice donor site contain a mutated "GT" motif. The mutated GT motif can inactivate splicing activity from both the major splice donor site and the adjacent cryptic splice donor site. An example of such a mutation is referred to herein as "MSD-2KO."

[0290] In one embodiment, the splice donor region may comprise the following sequence: CAGACA (SEQ ID NO: 5).

[0291] For example, in one embodiment, the mutated splice donor region may comprise the following sequence: GGCGACTGCAGACAACGCC (SEQ ID NO: 6).

[0292] Another example of an inactivating mutation is referred to herein as "MSD-2KOv2."

[0293] In one embodiment, the mutated splice donor region may comprise the following sequence: GTGGAGACT (SEQ ID NO: 7).

[0294] For example, in one embodiment, the mutated splice donor region may comprise the following sequence: GGCGAGTGGAGACTACGCC (SEQ ID NO: 8).

[0295] For example, in one embodiment, the mutated splice donor region may comprise the following sequence: AAGGCAACAGATAAATATGCCTT (SEQ ID NO: 14).

[0296] In one embodiment, the stem-loop 2 region can be deleted from the splice donor region, resulting in inactivation of both the major splice donor site and the adjacent cryptic splice donor site. Such a deletion is referred to herein as "ΔSL2."

[0297] A variety of different types of mutations can be introduced into a nucleic acid sequence to inactivate the major splice donor site and adjacent cryptic splice donor sites.

[0298] In one embodiment, the mutation is a functional mutation that eliminates or suppresses splicing activity in the splice region. The nucleotide sequences described herein can contain mutations or deletions in any of the nucleotides in SEQ ID NOs: 1, 3, 4, 9, 10 and / or 13. Suitable mutations are known to those of skill in the art and are described herein.

[0299] For example, a point mutation can be introduced into a nucleic acid sequence. As used herein, the term "point mutation" refers to any change to a single nucleotide. Point mutations include, for example, deletions, transitions, and transversions, which, when present in a protein-coding sequence, can be classified as nonsense mutations, missense mutations, or silent mutations. A "nonsense" mutation generates a stop codon. A "missense" mutation generates a codon that encodes a different amino acid. A "silent" mutation generates a codon that encodes either the same amino acid or a different amino acid that does not change the function of the protein. One or more point mutations can be introduced into a nucleic acid sequence containing a potential splice donor site. For example, a nucleic acid sequence containing a potential splice site can be mutated by introducing two or more point mutations into it.

[0300] To achieve attenuation of splicing from the splice donor region, at least two point mutations can be introduced at several positions within the nucleic acid sequence, including the major splice donor and potential splice donor sites. In one embodiment, the mutations can be within four nucleotides of the splice donor cleavage site, which in the canonical splice donor consensus sequence is A 1 G 2 / G 3 T 4 where " / " is the cleavage site.

[0301] It is well known in the art that splice donor cleavage sites can deviate from this consensus, particularly in viral genomes where other constraints, such as secondary structures within the vRNA packaging region, influence the same sequence. 3 T 4 Dinucleotides are generally known to be the least variable sequences within the canonical splice donor consensus sequence, and G 3 and / or T 4For example, in the case of the major splice donor site in the HIV-1 viral vector genome, this would be T 1 G 2 / G 3 T 4 (where " / " is the cleavage site). For example, in the case of a potential splice donor site in the HIV-1 viral vector genome, this can be G 1 A 2 / G 3 T 4 (where " / " is the cleavage site). Point mutations can also be introduced next to the splice donor site. For example, point mutations can be introduced upstream or downstream of the splice donor site. In embodiments in which a nucleic acid sequence containing a major and / or cryptic splice donor site is mutated by introducing multiple point mutations, point mutations can be introduced upstream and / or downstream of the cryptic splice donor site.

[0302] Construction of splice site mutants The splice site mutants of the invention can be constructed using a variety of techniques. For example, mutations can be introduced at specific loci by synthesizing oligonucleotides containing the mutant sequence flanked by restriction sites that allow ligation to fragments of the native sequence. After ligation, the resulting reconstructed sequence contains a derivative with the desired nucleotide insertion, substitution, or deletion.

[0303] Other known techniques that allow for the modification of DNA sequences include recombination approaches such as Gibson assembly, Golden-gate cloning, and in-fusion.

[0304] Alternatively, oligonucleotide-directed site-directed (or segment-directed) mutagenesis can be used to generate modified sequences with the required substitutions, deletions, or insertions. Deletion or truncation derivatives of splice site mutants can also be constructed by utilizing convenient restriction endonuclease sites flanking the desired deletion.

[0305] After restriction, the overhangs can be filled in and the DNA religated.

[0306] Exemplary methods for making such modifications are disclosed in Sambrook et al. (Molecular cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, 1989).

[0307] Splice site mutants can also be constructed by using techniques of PCR mutagenesis, chemical mutagenesis, chemical mutagenesis by forced nucleotide misincorporation (e.g., Liao and Wise, 1990), chemical mutagenesis (Drinkwater and Klinedinst, 1986), or by the use of random mutagenic oligonucleotides (Horwitz et al., 1989).

[0308] The present invention also provides (i) providing a nucleotide sequence encoding the RNA genome of a lentiviral vector described herein; and (ii) mutating the major splice donor site and the cryptic splice donor sites described herein in said nucleotide sequence. The present invention provides a method for producing a lentiviral vector nucleotide sequence, comprising:

[0309] Lentiviral vectors Lentiviruses are part of a larger group of retroviruses. A detailed list of lentiviruses can be found in Coffin et al. (1997) "Retroviruses," Cold Spring Harbor Laboratory Press, Eds: JM Coffin, SM Hughes, HE Varmus, pp. 758-763. Briefly, lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to, human immunodeficiency virus (HIV), the causative agent of human autoimmune deficiency syndrome (AIDS), and simian immunodeficiency virus (SIV). The non-primate lentivirus group includes the prototype "slow virus" Visna / Maedi virus (VMV), as well as the related Caprine Arthritis-Encephalitis Virus (CAEV), Equine Infectious Anemia Virus (EIAV), Feline Immunodeficiency Virus (FIV), Maedi-Visna Virus (MVV), and Bovine Immunodeficiency Virus (BIV). In one embodiment, the lentiviral vector is derived from an HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or visna lentivirus.

[0310] In one embodiment, the lentiviral vector is derived from HIV-1.

[0311] In one embodiment, the lentiviral vector is derived from HIV-2.

[0312] In one embodiment, the lentiviral vector is derived from EIAV.

[0313] In one embodiment, the lentiviral vector is derived from SIV.

[0314] In one embodiment, the lentiviral vector is derived from FIV.

[0315] In one embodiment, the lentiviral vector is derived from BIV.

[0316] In one embodiment, the lentiviral vector is derived from CAEV.

[0317] In one embodiment, the lentiviral vector is derived from a visna lentivirus.

[0318] The lentivirus family differs from retroviruses in that lentiviruses have the ability to infect both dividing and non-dividing cells (Lewis et al. (1992) EMBO J 11(8):3053-3058 and Lewis and Emerman (1994) J Virol 68(1):510-516). In contrast, other retroviruses, such as MLV, cannot infect non-dividing or slowly dividing cells (e.g., cells that make up muscle, brain, lung, and liver tissue).

[0319] As used herein, a lentiviral vector is a vector which comprises at least one component moiety derivable from a lentivirus, preferably which component moiety is involved in the biological mechanism by which the vector infects or transduces target cells and expresses the NOI.

[0320] Lentiviral vectors can be used to replicate the NOI in compatible target cells in vitro. Thus, described herein is a method for producing proteins in vitro by introducing a vector of the present invention into compatible target cells in vitro and growing the target cells under conditions that result in expression of the NOI. Proteins and NOIs can be recovered from the target cells by methods well known in the art. Suitable target cells include mammalian cell lines and other eukaryotic cell lines.

[0321] In some embodiments, the vector may have an "insulator" (a genetic sequence that acts as a barrier to block interactions between promoters and enhancers and reduce read-through from adjacent genes).

[0322] In one embodiment, an insulator is present between one or more lentiviral nucleic acid sequences to prevent promoter interference and read-through from adjacent genes. When an insulator is present in a vector between one or more lentiviral nucleic acid sequences, each of these isolated genes can be arranged as an individual expression unit.

[0323] The basic structure of retrovirus and lentivirus genomes has many common features, such as a 5'LTR and a 3'LTR (between or within which a packaging signal is located that enables the genome to be packaged), a primer binding site, an integration site that enables integration into the target cell genome, and the gag / pol and env genes that encode packaging components, which are polypeptides necessary for the assembly of viral particles. Lentiviruses have additional features, such as the rev gene and RRE sequence of HIV, which enable efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of infected target cells.

[0324] In the provirus, these genes are flanked on both sides by regions called long terminal repeats (LTRs). The LTRs direct the integration and transcription of the provirus. The LTRs can also function as enhancer-promoter sequences, controlling the expression of viral genes.

[0325] LTRs themselves are identical sequences and can be divided into three elements called U3, R, and U5. U3 is derived from a sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA, and U5 is derived from a sequence unique to the 5' end of the RNA. The sizes of these three elements can vary greatly depending on the retrovirus.

[0326] In typical retroviral vectors described herein, at least a portion of one or more protein coding regions essential for replication can be removed from the virus. For example, gag / pol and env can be absent or non-functional. This renders the viral vector replication-deficient.

[0327] Lentiviral vectors can be derived from primate lentiviruses (eg, HIV-1) or non-primate lentiviruses (eg, EIAV).

[0328] In general, a typical retroviral vector production system involves the separation of the viral genome from the essential viral packaging functions; these components are usually provided to the producer cell on separate DNA expression cassettes (alternatively known as plasmids, expression plasmids, DNA constructs, or expression constructs).

[0329] The vector genome contains the NOI. A vector genome typically requires a packaging signal (ψ), an internal expression cassette carrying the NOI, and (optionally) a post-transcriptional element (PRE), typically a central polypurine tract (cppt), a 3'-ppu, and a self-inactivating (SIN) LTR. The R-U5 region is required for proper polyadenylation of both the vector genome RNA and the NOI mRNA, as well as the process of reverse transcription. Optionally, the vector genome can contain an open reading frame, as described in WO 2003 / 064665, which allows for vector production in the absence of rev.

[0330] Packaging functions include the gag / pol and env genes, which are required for production of vector particles by producer cells. Addition of these functions in trans to the genome facilitates production of replication-deficient virus.

[0331] Gamma-retroviral vector production systems are typically three-component systems requiring a genome, gag / pol, and env expression constructs. HIV-1-based lentiviral vector production systems additionally require the accessory gene rev to be provided and for the vector genome to contain a rev response element (RRE). EIAV-based lentiviral vectors do not require rev to be provided in trans if an open reading frame (ORF) is present in the genome (see WO 2003 / 064665).

[0332] Typically, both the "external" promoter (which drives the vector genome cassette) and the "internal" promoter (which drives the NOI cassette) encoded within the vector genome cassette are strong eukaryotic or viral promoters, as are those driving other vector system components. Examples of such promoters include the CMV, EF1a, PGK, CAG, TK, SV40, and ubiquitin promoters. Strong "synthetic" promoters, such as promoters generated by DNA libraries (e.g., the JeT promoter), can also be used to drive transcription. Alternatively, tissue-specific promoters can be used to drive transcription, such as, for example, rhodopsin (Rho), rhodopsin kinase (RhoK), cone-rod homeobox-containing gene (CRX), neural retina-specific leucine zipper protein (NRL), vitelloid macular dystrophy 2 (VMD2), tyrosine hydroxylase, neuron-specific enolase (NSE) promoter, astrocyte-specific glial fibrillary acidic protein (GFAP) promoter, human α1-antitrypsin (hAAT) promoter, phosphoenolpyruvate carboxykinase (PE) promoter, and the like. PCK), liver fatty acid binding protein promoter, Flt-1 promoter, INF-β promoter, Mb promoter, SP-B promoter, SYN1 promoter, WASP promoter, SV40 / hAlb promoter, SV40 / CD43, SV40 / CD45, NSE / RU5' promoter, ICAM-2 promoter, GPIIb promoter, GFAP promoter, fibronectin promoter, endoglin promoter, elastase-1 promoter, desmin promoter, CD68 promoter, CD14 promoter, and B29 promoter.

[0333] The production of retroviral vectors involves transient co-transfection of producer cells with these DNA components or the use of stable producer cell lines, in which all of the components are stably integrated into the producer cell genome (e.g., Stewart HJ, Fong-Wong L, Strickland I, Chipchase D, Kelleher M, Stevenson L, Thoree V, McCarthy J, Ralph GS, Mitrophanous KA and Radcliffe PA. (2011). Hum Gene Ther. Mar; 22 (3): 357-69). Another approach is to use stable packaging cells (in which the packaging components are stably integrated), and then, if necessary, transiently transfect with a vector genome plasmid (e.g., Stewart, HJ, MALeroux-Carlucci, CJ Syon, KA Mitrophanous and PA Radcliffe (2009). Gene Ther. Jun; 16 (6): 805-14). It is also possible to generate alternative, less complete packaging cell lines (in which only one or two packaging components are stably integrated into the cell line) and transiently transfect the defective components to generate vectors. Producer cells can also express regulatory proteins, such as members of the tet repressor (TetR) protein group of transcriptional regulators (e.g., T-Rex, Tet-On, and Tet-Off), members of the coumarate-inducible switch system group of transcriptional regulators (e.g., coumarate repressor (CymR) protein), or RNA-binding proteins (e.g., TRAP - tryptophan-activated RNA-binding protein).

[0334] In one embodiment of the present invention, the viral vector is derived from EIAV. EIAV has the simplest genomic structure of lentiviruses and is particularly preferred for use in the present invention. In addition to the gag / pol and env genes, EIAV encodes three other genes: tat, rev, and S2. Tat acts as a transcriptional activator of the viral LTR (Derse and Newbold (1993) Virology 194(2):530-536 and Maury et al. (1994) Virology 200(2):632-642), and rev regulates and integrates viral gene expression via the rev response element (RRE) (Martarano et al. (1994) J Virol 68(5):3102-3111). The mechanisms of action of these two proteins are thought to be broadly similar to those in primate viruses (Martarano et al. (1994) J Virol 68(5):3102-3111). The function of S2 is unknown. Additionally, the EIAV protein Ttm has been identified, which is encoded by the first exon of tat, which is spliced ​​into the env coding sequence at the start of the transmembrane protein. In another embodiment of the present invention, the viral vector is derived from HIV. HIV differs from EIAV in that it does not encode S2, but, unlike EIAV, it encodes vif, vpr, vpu, and nef.

[0335] The term "recombinant retroviral or lentiviral vector" (RRV) refers to a vector that carries sufficient retroviral genetic information to allow packaging of the RNA genome into viral particles that can transduce target cells in the presence of packaging components. Transduction of target cells can involve reverse transcription and integration into the target cell genome. RRVs carry non-viral coding sequences to be delivered to target cells by the vector. RRVs are incapable of autonomous (independent) replication to produce infectious retroviral particles in target cells. RRVs usually lack functional gag / pol and / or env genes, and / or other genes essential for replication.

[0336] Preferably, the RRV vectors of the invention have a minimal viral genome.

[0337] As used herein, the term "minimal viral genome" means that a viral vector has been engineered to remove non-essential elements while retaining essential elements to confer the necessary functionality for infection, transduction, and delivery of an NOI to a target cell. Further details of this methodology can be found in WO 1998 / 17815 and WO 99 / 32646. Minimal EIAV vectors lack the tat, S2 genes, and optionally, rev, none of which are provided in trans in the production system. Minimal HIV vectors lack vif, vpr, vpu, tat, and nef.

[0338] The expression plasmid used to produce the vector genome in producer cells contains transcriptional regulatory control sequences operably linked to the retroviral genome to direct transcription of the genome in producer / packaging cells. All third-generation lentiviral vectors have a deletion in the 5' U3 enhancer-promoter region, allowing transcription of the vector genome RNA to be driven by a heterologous promoter, such as another viral promoter, e.g., the CMV promoter (described below). This feature allows for tat-independent vector production. Some lentiviral vector genomes require additional sequences for efficient virus production. For example, an RRE sequence may be included, particularly in the case of HIV. However, the requirement for the RRE in the (separate) GagPol cassette (and the dependency on rev, conferred in trans) can be reduced or eliminated by codon optimization of the GagPol ORF. Further details of this method can be found in WO 2001 / 79518.

[0339] Alternative sequences that perform the same function as the rev / RRE system are also known. For example, a functional analog of the rev / RRE system is found in the Mason-Pfizer monkey virus. This contains an RRE-type sequence within its genome known as a constitutive transport element (CTE), which is thought to interact with a factor within infected cells. The cellular factor may be considered a rev analog. Thus, a CTE may be used as a substitute for the rev / RRE system. Any other functional equivalent of the Rev protein that is known or becomes available may be relevant to the present invention. For example, it is known that the Rex protein of HTLV-1 can functionally replace the Rev protein of HIV-1. Rev and RRE may be absent or non-functional in vectors for use in the methods of the present invention, or rev and RRE, or functionally equivalent systems, may be present.

[0340] As used herein, the term "functional substitute" refers to a protein or sequence having an alternative sequence that performs the same function as another protein or sequence. The term "functional substitute" is used interchangeably herein with "functional equivalent" and "functional analog," which have the same meaning.

[0341] SIN vector The lentiviral vectors described herein can be used in a self-inactivating (SIN) configuration, in which viral enhancer and promoter sequences are deleted. SIN vectors can be manufactured and used to transduce non-dividing target cells in vivo, ex vivo, or in vitro with efficacies similar to those of non-SIN vectors. Transcriptional inactivation of the long terminal repeats (LTRs) in SIN proviruses should prevent vRNA recruitment, a feature that further reduces the likelihood of replication-competent virus formation. This should also allow for regulated expression of genes from internal promoters by eliminating the cis-acting effects of the LTRs.

[0342] For example, self-inactivating retroviral vector systems have been constructed by deleting transcriptional enhancers or enhancers and promoters (within the U3 region of the 3' LTR). After rounds of vector reverse transcription and integration, these changes are copied into both the 5' and 3' LTRs, resulting in a transcriptionally inactive provirus. However, any promoters internal to the LTR in such vectors will still be transcriptionally active. This strategy has been used to eliminate the effects of enhancers and promoters in the viral LTR on transcription from internally located genes. Such effects include enhanced transcription or repressed transcription. This strategy can also be used to eliminate downstream transcription from the 3' LTR into genomic DNA. This is of particular interest in human gene therapy, where it is important to prevent the inadvertent activation of any endogenous oncogenes. Yu et al. (1986) PNAS 83:3194-98; Marty et al. (1990) Biochimie 72:885-7; Naviaux et al. (1996) J. Virol. 70:5701-5; Iwakuma et al. (1999) Virol. 261:120-32; Deglon et al. (2000) Human Gene Therapy 11:179-90. SIN lentiviral vectors are described in US 6,924,123 and US 7,056,699.

[0343] Replication-deficient lentiviral vectors In the genome of a replication-defective lentiviral vector, the gag / pol and / or env sequences can be mutated and / or non-functional.

[0344] In typical lentiviral vectors described herein, at least a portion of one or more coding regions for proteins essential for viral replication may be removed from the vector, rendering the viral vector replication-deficient. Portions of the viral genome may also be replaced with an NOI to generate vectors containing an NOI that are capable of transducing non-dividing target cells and / or integrating the viral genome into the target cell genome.

[0345] In one embodiment, the lentiviral vector is a non-integrating vector as described in WO 2006 / 010834 and WO 2007 / 071994.

[0346] In another embodiment, the vector is capable of delivering defective or missing viral RNA sequences. In another embodiment, a heterologous binding domain (heterologous to gag) located on the RNA to be delivered and a cognate binding domain on Gag or GagPol can be used to ensure packaging of the RNA to be delivered. Both of these vectors are described in WO 2007 / 072056.

[0347] NOIs and polynucleotides The polynucleotides of the present invention may comprise DNA or RNA. They may be single-stranded or double-stranded. It will be understood by those skilled in the art that, as a result of the degeneracy of the genetic code, many different polynucleotides may encode the same polypeptide. It should also be understood that those skilled in the art may, using conventional techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the present invention to reflect the codon usage of any particular host organism in which the polypeptide of the present invention will be expressed.

[0348] The polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or lifespan of polynucleotides of the invention.

[0349] Polynucleotides, such as DNA polynucleotides, may be produced by recombinant methods, synthetic methods, or any means available to those of skill in the art. They may also be cloned by standard techniques.

[0350] Longer polynucleotides are generally produced using recombinant means, for example, polymerase chain reaction (PCR) cloning techniques. This involves creating a pair of primers (e.g., about 15-30 nucleotides) that flank the target sequence to be cloned, contacting the primers with mRNA or cDNA obtained from animal or human cells, performing PCR under conditions that result in amplification of the desired region, isolating the amplified fragment (e.g., by purifying the reaction mixture on an agarose gel), and recovering the amplified DNA. Primers can be designed to contain appropriate restriction enzyme recognition sites so that the amplified DNA can be cloned into an appropriate vector.

[0351] Common retroviral vector elements Promoters and enhancers Expression of the NOI and polynucleotides may be controlled using control sequences, such as transcriptional regulatory elements or translational repression elements, including promoters, enhancers and other expression regulation signals (e.g., the tet repressor (TetR) system) or Transgene Repression In vector Production cell system (TRIP) or other regulatory elements of the NOI described herein.

[0352] Prokaryotic promoters and promoters that function in eukaryotic cells can be used. Tissue-specific or stimulus-specific promoters can be used. Chimeric promoters containing sequence elements from two or more different promoters can also be used.

[0353] Suitable promoter sequences are strong promoters, including those derived from the genomes of viruses such as polyomavirus, adenovirus, fowlpox virus, bovine papillomavirus, avian sarcoma virus, cytomegalovirus (CMV), retroviruses and simian virus 40 (SV40), or heterologous mammalian promoters such as the actin promoter, EF1a, CAG, TK, SV40, ubiquitin, PGK or ribosomal protein promoters. Alternatively, tissue-specific promoters can be used to drive transcription, such as, for example, rhodopsin (Rho), rhodopsin kinase (RhoK), cone-rod homeobox-containing gene (CRX), neural retina-specific leucine zipper protein (NRL), vitelloid macular dystrophy 2 (VMD2), tyrosine hydroxylase, neuron-specific enolase (NSE) promoter, astrocyte-specific glial fibrillary acidic protein (GFAP) promoter, human α1-antitrypsin (hAAT) promoter, phosphoenolpyruvate carboxykinase (PE) promoter, and the like. PCK), liver fatty acid binding protein promoter, Flt-1 promoter, INF-β promoter, Mb promoter, SP-B promoter, SYN1 promoter, WASP promoter, SV40 / hAlb promoter, SV40 / CD43, SV40 / CD45, NSE / RU5' promoter, ICAM-2 promoter, GPIIb promoter, GFAP promoter, fibronectin promoter, endoglin promoter, elastase-1 promoter, desmin promoter, CD68 promoter, CD14 promoter, and B29 promoter.

[0354] Transcription of the NOI can be further enhanced by inserting an enhancer sequence into the vector. Enhancers are relatively orientation- and position-independent, but enhancers from eukaryotic viruses, such as the SV40 enhancer and the CMV early promoter enhancer, can be used. The enhancer can be spliced ​​into the vector at a position 5' or 3' to the promoter, but is preferably located at a site 5' from the promoter.

[0355] The promoter may further comprise features for ensuring or enhancing expression in appropriate target cells. For example, such features may be conserved regions, such as the Pribnow box or the TATA box. The promoter may contain other sequences that affect the level of expression of the nucleotide sequence (e.g., maintain, enhance, or reduce the level). Suitable other sequences include the Sh1 intron or the ADH intron. Other sequences include inducible elements, such as elements inducible by temperature, chemicals, light, or stress. Suitable elements for enhancing transcription or translation may also be present.

[0356] Regulators of NOIs A complicating factor in the generation of retroviral packaging / producer cell lines and retroviral vector production is that constitutive expression of certain retroviral vector components and NOIs is cytotoxic, leading to the death of cells expressing these components and thus preventing vector production. Therefore, expression of these components (e.g., gag-pol and envelope proteins such as VSV-G) can be regulated. Expression of other non-cytotoxic vector components, such as rev, can also be regulated to minimize metabolic burden on cells. Thus, the modular constructs or nucleotide sequences encoding vector components and / or cells described herein can contain cytotoxic and / or non-cytotoxic vector components associated with at least one regulatory element. As used herein, the term "regulatory element" refers to any element that can affect (enhance or decrease) the expression of an associated gene or protein. Regulatory elements include gene switch systems, transcriptional regulatory elements, and translational repression elements.

[0357] Numerous prokaryotic regulatory systems have been employed to generate gene switches in mammalian cells. Many retroviral packaging and producer cell lines are controlled using gene switch systems (e.g., tetracycline and coumarate-inducible switch systems), thus allowing the expression of one or more retroviral vector components to be switched on during vector production. Gene switch systems include those involving the TetR protein group of transcriptional regulators (e.g., T-Rex, Tet-On, and Tet-Off), those involving the coumarate-inducible switch system group of transcriptional regulators (e.g., CymR protein), and those involving RNA-binding proteins (e.g., TRAP).

[0358] One such tetracycline-inducible system is the tetracycline repressor (TetR) system, which is based on the T-REx™ system. For example, in such a system, the tetracycline operator (TetO2) is positioned so that its first nucleotide is 10 bp from the 3' end of the last nucleotide of the TATATAA element of the human cytomegalovirus major immediate-early promoter (hCMVp), in which case TetR can function alone as a repressor (Yao F, Svensjo T, Winkler T, Lu M, Eriksson C, Eriksson E., 1998, Hum Gene Ther;9:1939-1950). In such a system, expression of an NOI can be controlled by a CMV promoter into which two copies of the TetO2 sequence have been inserted in tandem. The TetR homodimer binds to the TetO2 sequence in the absence of inducer (tetracycline or its analog doxycycline [dox]) and physically blocks transcription from the upstream CMV promoter. When inducer is present, it binds to the TetR homodimer and causes an allosteric change so that it can no longer bind to the TetO2 sequence, resulting in gene expression. The TetR gene can be codon-optimized, as this has been shown to improve translation efficiency and result in tighter control of TetO2-controlled gene expression.

[0359] The TRAP system, described in WO 2015 / 092440, provides another method for suppressing expression of an NOI in producer cells during vector production. TRAP-binding sequence (e.g., TRAP-tbs) interactions form the basis of a transgene protein suppression system for retroviral vector production when a constitutive and / or strong promoter (including tissue-specific promoters) driving the transgene is desirable, particularly when expression of the transgene protein in producer cells results in reduced vector titer and / or when viral vector delivery of the transgene-derived protein induces an immune response in vivo (Maunder et al., Nat Commun. (2017) Mar 27;8).

[0360] Briefly, the TRAP-tbs interaction results in a translational block, suppressing the translation of transgene proteins (Maunder et al., Nat Commun. (2017) Mar 27;8). Translational blockade is only effective in producer cells and does not, by itself, interfere with DNA- or RNA-based vector systems. The TRiP system can suppress translation when transgene proteins are expressed from constitutive and / or strong promoters (including tissue-specific promoters from mono- or polycistronic mRNAs). It has been demonstrated that unregulated expression of transgene proteins can reduce vector titers and affect the quality of vector products. Suppression of transgene proteins in both transient and stable PaCL / PCL vector production systems is beneficial for producer cells to prevent a decrease in vector titer, especially when toxicity or molecular burden issues lead to cellular stress, when viral vector delivery of transgene-derived proteins induces immune responses in vivo, when the use of gene-edited transgenes can result in on- or off-target effects, or when transgene proteins affect vector and / or envelope glycoprotein clearance.

[0361] Envelope and pseudotyping In a preferred embodiment, the viral vectors described herein are pseudotyped. In this regard, pseudotyping can provide one or more advantages. For example, the env gene product of HIV-based vectors will limit these vectors to infecting only cells that express a protein called CD4. However, if the env gene in these vectors is replaced with env sequences from other enveloped viruses, they may have a broader infectious spectrum (Verma and Somia (1997) Nature 389(6648):239-242). For example, researchers have pseudotyped HIV-based vectors with glycoproteins from VSV (Verma and Somia (1997) Nature 389(6648):239-242).

[0362] In another alternative, the Env protein can be a modified Env protein, such as a mutant or engineered Env protein. Modifications can be made or selected to introduce targeting capabilities, reduce toxicity, or for other purposes (Valsesia-Wittman et al. 1996 J Virol 70:2056-64; Nilson et al. (1996) Gene Ther 3(4):280-286; and Fielding et al. (1998) Blood 91(5):1802-1809 and references cited therein).

[0363] Vectors can be pseudotyped with any molecule of choice.

[0364] As used herein, "env" shall mean the endogenous lentiviral envelope or heterologous envelope described herein.

[0365] VSV-G The envelope glycoprotein (G) of the rhabdovirus vesicular stomatitis virus (VSV) is an envelope protein that has been shown to be capable of pseudotyping certain enveloped viruses and viral vector virions.

[0366] It was first shown by Emi et al. ((1991) Journal of Virology 65:1202-1207) that MoMLV-based retroviral vectors can be pseudotyped in the absence of retroviral envelope proteins. WO 1994 / 294440 teaches that retroviral vectors can be successfully pseudotyped with VSV-G. These pseudotyped VSV-G vectors can be used to transduce a wide range of mammalian cells. Subsequently, Abe et al. (1998) J Virol 72(8)6356-6361 teaches that non-infectious retroviral particles can be made infectious by the addition of VSV-G.

[0367] Burns et al. ((1993) Proc. Natl. Acad. Sci. USA 90:8033-7) successfully pseudotyped the retrovirus MLV with VSV-G, resulting in a vector with an altered host range compared to the native form of MLV. VSV-G pseudotyped vectors have been shown to infect not only mammalian cells but also cell lines derived from fish, reptiles, and insects (Burns et al. (1993) supra). They have also been shown to be more efficient than conventional amphotropic envelopes with respect to various cell lines (Yee et al. (1994) Proc. Natl. Acad. Sci. USA 91:9564-9568, Emi et al. (1991) Journal of Virology 65:1202-1207). The VSV-G protein can be used to pseudotype certain retroviruses. This is because the cytoplasmic tail can interact with the retroviral core.

[0368] The provision of a non-retroviral pseudotyping envelope, such as the VSV-G protein, offers the advantage that vector particles can be concentrated to high titers without a loss of infectivity (Akkina et al. (1996) J. Virol. 70:2581-5). Retroviral envelope proteins appear unable to withstand the shear forces during ultracentrifugation, presumably because they consist of two non-covalently bound subunits. The interaction between these subunits can be disrupted by centrifugation. On the other hand, the VSV glycoprotein consists of a single unit. Therefore, VSV-G protein pseudotyping could potentially offer advantages for both efficient target cell infection / transduction during the manufacturing process.

[0369] WO 2000 / 52188 describes the generation of pseudotyped retroviral vectors from stable producer cell lines carrying the vesicular stomatitis virus G protein (VSV-G) as the membrane-associated viral envelope protein and provides the gene sequence for the VSV-G protein.

[0370] Ross River virus Ross River virus envelope has been used to pseudotype non-primate lentiviral vectors (FIV), which transduced primarily the liver after systemic administration (Kang et al., 2002, J. Virol., 76(18):9378-9388). The efficiency was reported to be 20-fold greater than that obtained with VSV-G pseudotyped vectors, and it did not cause significant cytotoxicity as measured by serum levels of liver enzymes, which would suggest hepatotoxicity.

[0371] Baculovirus GP64 The baculovirus GP64 protein has been shown to be an alternative to VSV-G for viral vectors used in the large-scale production of high-titer virus required for clinical and commercial applications (Kumar M, Bradow BP, Zimmerberg J (2003) Hum Gene Ther. 14(1):67-77). Compared to VSV-G pseudotyped vectors, GP64 pseudotyped vectors have similar broad tropism and similar native titers. Because expression of GP64 does not kill cells, HEK293T-based cell lines that constitutively express GP64 can be generated.

[0372] Alternative Envelopes Other envelopes that give reasonable titers when used to pseudotype EIAV include Mokola, rabies, Ebola, and LCMV (lymphocytic choriomeningitis virus). Intravenous injection of 4070A-pseudotyped lentivirus into mice resulted in maximal gene expression in the liver.

[0373] Packaging Sequence As used in the context of the present invention, the term "packaging signal" is also interchangeably referred to as "packaging sequence" or "psi" and is used in reference to a non-coding cis-acting sequence required for encapsidation of retroviral RNA strands during viral particle formation. In HIV-1, this sequence is located in a locus extending from upstream of the major splice donor site (SD) to at least the gag initiation codon (which may include some or all of the 5' sequence of gag up to nucleotide 688). In EIAV, the packaging signal includes the R region into the 5' coding region of Gag.

[0374] As used herein, the term "extended packaging signal" or "extended packaging sequence" refers to the use of sequences surrounding the psi sequence extended further into the gag gene. The inclusion of these additional packaging sequences can increase the efficiency of vector RNA insertion into viral particles.

[0375] Feline immunodeficiency virus (FIV) RNA encapsidation determinants have been shown to be discrete and discontinuous, comprising one region (R-U5) at the 5' end of the genomic mRNA and another region located within the proximal 311 nt of gag (Kaye et al., J Virol. Oct;69(10):6588-92 (1995)).

[0376] Internal ribosome entry site (IRES) Insertion of an IRES element allows expression of multiple coding regions from a single promoter (Adam et al., supra; Koo et al. (1992) Virology 186:669-675; Chen et al. 1993 J. Virol 67:2142-2148). IRES elements were first found in the non-translated 5' ends of picornaviruses, where they promote cap-independent translation of viral proteins (Jang et al. (1990) Enzyme 44:292-309). When located between open reading frames in an RNA, IRES elements facilitate ribosome entry at the IRES element and subsequent initiation of downstream translation, thereby enabling efficient translation of the downstream open reading frame.

[0377] A review of IRES is given in Mountford and Smith (TIG May 1995 vol 11, No 5:179-184). Many different IRES sequences are known, including those from encephalomyocarditis virus (EMCV) (Ghattas, IR et al., Mol. Cell. Biol., 11:5848-5859 (1991)); BiP protein [Macejak and Sarnow, Nature 353:91 (1991)]; the antennapedia gene (exons d and e) of Drosophila [Oh et al., Genes & Development, 6:1643-1653 (1992)], and in poliovirus (PV) [Pelletier and Sonenberg, Nature 334:320-325 (1988); see also Mountford and Smith, TIG 11, 179-184 (1985)].

[0378] IRES elements from PV, EMCV and swine vesicular disease virus have already been used in retroviral vectors (Coffin et al., supra).

[0379] The term "IRES" includes any sequence or combination of sequences that acts as an IRES or improves the function of an IRES. The IRES can be viral (e.g., the EMCV IRES, the PV IRES, or an FMDV 2A-like sequence) or cellular (e.g., the FGF2 IRES, the NRF IRES, the Notch 2 IRES, or the EIF4 IRES).

[0380] In order for the IRES to be able to initiate translation of each polynucleotide, it should be located between or before said polynucleotides in the modular construct.

[0381] Nucleotide sequences utilized for the development of stable cell lines require the addition of selectable markers for the selection of cells in which stable integration has occurred. These selectable markers can be expressed as single transcription units within the nucleotide sequence, or it may be preferable to use IRES elements to initiate translation of the selectable marker in a polycistronic message (Adam et al. 1991 J. Virol. 65, 4985).

[0382] Genetic orientation and insulators It is well known that nucleic acids have directionality (orientation), which ultimately affects mechanisms such as transcription and replication within a cell. Thus, genes can have a relative orientation to each other when they are part of the same nucleic acid construct.

[0383] In certain embodiments of the present invention, at least two nucleic acid sequences present at the same locus in a cell or construct may be present in opposite and / or alternate orientations. In other words, in certain embodiments of the present invention at this particular locus, no pair of consecutive genes has the same orientation. This may help prevent both transcriptional and translational read-through when the regions are expressed in the same physical location in a host cell.

[0384] Having alternate orientations provides benefits for retroviral vector production when the nucleic acids required for vector production are based at the same locus within the cell, and this in turn may improve the safety of the resulting construct in preventing the generation of replication-competent retroviral vectors.

[0385] Where nucleic acid sequences are present in reverse and / or alternate orientations, the use of insulators may prevent inappropriate expression or silencing of the NOI from its genetic environment.

[0386] The term "insulator" refers to a class of DNA sequence elements that have the ability to protect genes from surrounding regulatory signaling when bound to insulator-binding proteins. There are two types of insulators: enhancer-blocking and chromatin barrier. When an insulator is located between a promoter and an enhancer, the enhancer-blocking function of the insulator protects the promoter from the transcriptional enhancing effects of the enhancer (Geyer and Corces 1992; Kellum and Schedl 1992). Chromatin barrier insulators function by preventing the progression of nearby condensed chromatin, which converts transcriptionally active chromatin regions into transcriptionally inactive chromatin regions, resulting in the silencing of gene expression. Insulators, which inhibit heterochromatin spreading and gene silencing, recruit enzymes involved in histone modification to disrupt this process (Yang J, Corces VG. 2011;110:43-76; Huang, Li et al. 2007; Dhillon, Raab et al. 2009). Insulators can have one or both of these functions. The chicken β-globin insulator (cHS4) is one such example. This insulator, the most widely studied vertebrate insulator, is highly G+C-rich and functions as both an enhancer blocker and a heterochromatin barrier (Chung JH, Whitely M, Felsenfeld G. Cell. 1993;74:505-514). Other such insulators with enhancer-blocking function include, but are not limited to, human β-globin insulator 5 (HS5), human β-globin insulator 1 (HS1), and chicken β-globin insulator (cHS3) (Farrell CM1, West AG, Felsenfeld G., Mol Cell Biol. 2002 Jun;22(11):3820-31; J Ellis et al. EMBO J. 1996 Feb 1;15(3):562-568).In addition to reducing undesired distal interactions, insulators can also help prevent promoter interference between adjacent retroviral nucleic acid sequences (i.e., where a promoter from one transcription unit impairs expression of an adjacent transcription unit). If insulators are used between each of the retroviral vector nucleic acid sequences, the reduction in direct readthrough will help prevent the formation of replication-competent retroviral vector particles.

[0387] An insulator may be present between each of the retroviral nucleic acid sequences, in one embodiment the use of an insulator prevents promoter-enhancer interactions and prevents one NOI expression cassette from interacting with another NOI expression cassette in the nucleotide sequences encoding the vector components.

[0388] An insulator element can be present between the vector genome sequence and the gag-pol sequence. This therefore limits the possibility of producing "wild-type" such as replicative retroviral vectors and RNA transcripts, improving the safety profile of the construct. The use of insulator elements to improve expression of stably integrated multigene vectors is cited in Moriarity et al., Nucleic Acids Res. 2013 Apr;41(8):e92.

[0389] Vector titer Those skilled in the art will appreciate that there are many different methods for determining the titer of a lentiviral vector. Titer is often expressed as transducing units / mL (TU / mL). Titer can be increased by increasing the number of vector particles and by increasing the specific activity of the vector preparation.

[0390] therapeutic use The lentiviral vectors described herein or cells or tissues transduced with the lentiviral vectors described herein may be used in medicine.

[0391] Additionally, the lentiviral vectors described herein, the producer cells of the invention, or cells or tissues transduced with the lentiviral vectors described herein can be used in the manufacture of a medicament to deliver a nucleotide of interest to a target site where it is required. Such use of the lentiviral vectors or transduced cells of the invention can be for therapeutic or diagnostic purposes, as previously described.

[0392] Accordingly, there are provided cells transduced with the lentiviral vectors described herein.

[0393] "Cells transduced by a viral vector particle" should be understood as cells, particularly target cells, into which the nucleic acid carried by the viral vector particle has been introduced.

[0394] In a preferred embodiment, the nucleotide of interest provides a therapeutic effect.

[0395] "Target cells" should be understood as cells in which it is desired to express the NOI. The NOI may be introduced into the target cells using the viral vector of the present invention. Delivery to the target cells may be performed in vivo, ex vivo or in vitro.

[0396] The NOI may have therapeutic or diagnostic uses. Suitable NOIs include, but are not limited to, sequences encoding enzymes, cofactors, cytokines, chemokines, hormones, antibodies, antioxidant molecules, engineered immunoglobulin-like molecules, single-chain antibodies, fusion proteins, immune co-stimulatory molecules, immunomodulatory molecules, chimeric antigen receptors, trans-domain negative mutants of target proteins, toxins, conditional toxins, antigens, transcription factors, structural proteins, reporter proteins, subcellular localization signals, tumor suppressor proteins, growth factors, membrane proteins, receptors, vasoactive proteins and peptides, antiviral proteins and ribozymes, and derivatives thereof (e.g., derivatives with associated reporter groups). The NOI may also encode microRNAs. Without wishing to be bound by theory, it is believed that microRNA processing is inhibited by TRAP.

[0397] In one embodiment, the NOI may be useful in the treatment of neurodegenerative disorders.

[0398] In another embodiment, the NOI may be useful in the treatment of Parkinson's disease.

[0399] In another embodiment, the NOI may encode one or more enzymes involved in dopamine synthesis. For example, the enzymes may be one or more of the following: tyrosine hydroxylase, GTP-cyclohydrolase I and / or aromatic amino acid dopa decarboxylase. The sequences of all three genes are available (GenBank® accession numbers X05290, U19523 and M76180 respectively).

[0400] In another embodiment, the NOI may encode vesicular monoamine transporter 2 (VMAT2). In another embodiment, the viral genome may comprise an NOI encoding the aromatic amino acid dopa decarboxylase and an NOI encoding VMAT2. Such a genome may be used in the treatment of Parkinson's disease, particularly in conjunction with peripheral administration of L-DOPA.

[0401] In another embodiment, the NOI may encode a therapeutic protein or a combination of therapeutic proteins.

[0402] In another embodiment the NOI may encode one or more proteins selected from the group consisting of glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), neurotrophin-3 (NT-3), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), interleukin 1 beta (IL-1β), tumour necrosis factor alpha (TNF-α), insulin growth factor-2, VEGF-A, VEGF-B, VEGF-C / VEGF-2, VEGF-D, VEGF-E, PDGF-A, PDGF-B, heterodimers and homodimers of PDFG-A and PDFG-B.

[0403] In another embodiment, the NOI may encode one or more anti-angiogenic proteins selected from the group consisting of angiostatin, endostatin, platelet factor 4, pigment epithelium-derived factor (PEDF), placental growth factor, restin, interferon-alpha, interferon-inducible protein, gro-beta and tubedown-1, interleukin (IL)-1, IL-12, retinoic acid, anti-VEGF antibodies or fragments / variants thereof such as aflibercept, thrombospondin, VEGF receptor proteins such as those described in US 5,952,199 and US 6,100,071, and anti-VEGF receptor antibodies.

[0404] In another embodiment the NOI may encode an anti-inflammatory protein, antibody or fragment / variant of a protein or antibody selected from the group consisting of NF-kB inhibitor, IL1 beta inhibitor, TGF beta inhibitor, IL-6 inhibitor, IL-23 inhibitor, IL-18 inhibitor, tumour necrosis factor alpha and beta, lymphotoxin alpha and beta, LIGHT inhibitor, alpha synuclein inhibitor, tau inhibitor, beta amyloid inhibitor, IL-17 inhibitor.

[0405] In another embodiment, the NOI may encode the cystic fibrosis transmembrane conductance regulator (CFTR).

[0406] In another embodiment, the NOI may encode a protein that is normally expressed in ocular cells.

[0407] In another embodiment, the NOI may encode a protein that is normally expressed in photoreceptor cells and / or retinal pigment epithelial cells.

[0408] In a further embodiment the NOI may encode a protein selected from the group including RPE65, aryl hydrocarbon interacting receptor protein-like 1 (AIPL1), CRB1, lecithin retinal acetyltransferase (LRAT), photoreceptor-specific homeobox (CRX), retinal guanylate cyclase (GUCY2D), RPGR-interacting protein 1 (RPGRIP1), LCA2, LCA3, LCA5, dystrophin, PRPH2, CNTF, ABCR / ABCA4, EMP1, TIMP3, MERTK, ELOVL4, MYO7A, USH2A, VMD2, RLBP1, COX-2, FPR, harmonin, Rab escort protein 1, CNGB2, CNGA3, CEP 290, RPGR, RS1, RP1, PRELP, glutathione pathway enzymes and opticin.

[0409] In another embodiment the NOI may encode human clotting factors VIII or IX.

[0410] In other embodiments, the NOI is selected from the group consisting of phenylalanine hydroxylase (PAH), methylmalonyl-CoA mutase, propionyl-CoA carboxylase, isovaleryl-CoA dehydrogenase, branched chain keto acid dehydrogenase complex, glutaryl-CoA dehydrogenase, acetyl-CoA carboxylase, propionyl-CoA carboxylase, 3-methylcrotonyl-CoA carboxylase, pyruvate carboxylase, carbamoyl-phosphate synthase ammonia, ornithine transcarbamylase, glucosylceramidase beta, alpha-galactosidase A, glucosylceramidase The gene may encode one or more proteins involved in metabolism selected from the group including beta, cystinosine, glucosamine (N-acetyl)-6-sulfatase, N-acetyl-alpha-glucosaminidase, N-sulfoglucosamine sulfohydrolase, galactosamine-6-sulfatase, arylsulfatase A, cytochrome B-245 beta, ABCD1, ornithine carbamoyltransferase, argininosuccinate synthase, argininosuccinate lysase, arginase 1, alanine glycoxylate aminotransferase, ATP-binding cassette, and subfamily B members.

[0411] In other embodiments the NOI may encode a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In one embodiment the CAR is an anti-5T4 CAR. In other embodiments the NOI may encode B cell maturation antigen (BCMA), CD19, CD22, CD20, CD47, CD138, CD30, CD33, CD123, CD70, prostate specific membrane antigen (PSMA), Lewis Y antigen (LeY), tyrosine protein kinase transmembrane receptor (ROR1), mucin 1, cell surface associated (Muc1), epithelial cell adhesion molecule (EpCAM), endothelial growth factor receptor (EGFR), insulin, protein tyrosine phosphatase, non-receptor type 22, interleukin 2 receptor alpha, helicase C domain 1 induced interferon, human epidermal growth factor receptor (HER2), glypican 3 (GPC3), disialoganglioside (GD2), mesiothelin, vascular endothelial growth factor receptor 2 (VEGFR2).

[0412] In one embodiment the NOI encodes BCMA.

[0413] In one embodiment the NOI encodes CD19.

[0414] In one embodiment the NOI encodes CD22.

[0415] In one embodiment the NOI encodes CD20.

[0416] In one embodiment the NOI encodes CD47.

[0417] In one embodiment the NOI encodes CD138.

[0418] In one embodiment the NOI encodes CD30.

[0419] In one embodiment the NOI encodes CD33.

[0420] In one embodiment the NOI encodes CD123.

[0421] In one embodiment the NOI encodes CD70.

[0422] In one embodiment the NOI encodes PSMA.

[0423] In one embodiment the NOI encodes LeY.

[0424] In one embodiment the NOI encodes ROR1.

[0425] In one embodiment the NOI encodes mucin1.

[0426] In one embodiment the NOI encodes Mucl.

[0427] In one embodiment the NOI encodes EpCAM.

[0428] In one embodiment the NOI encodes EGFR.

[0429] In one embodiment the NOI encodes insulin.

[0430] In one embodiment the NOI encodes a protein tyrosine phosphatase.

[0431] In one embodiment the NOI encodes non-receptor type 22.

[0432] In one embodiment the NOI encodes the interleukin 2 receptor alpha.

[0433] In one embodiment the NOI encodes a helicase C domain 1 induced interferon.

[0434] In one embodiment the NOI encodes HER2.

[0435] In one embodiment the NOI encodes GPC3.

[0436] In one embodiment the NOI encodes GD2.

[0437] In one embodiment the NOI encodes mesiothelin.

[0438] In one embodiment the NOI encodes VEGFR2.

[0439] In another embodiment the NOI may encode a chimeric antigen receptor (CAR) against an NKG2D ligand selected from the group including ULBP1, 2 and 3, H60, Rae-1a, b, g, d, MICA, MICB.

[0440] In a further embodiment the NOI is selected from the group consisting of SGSH, SUMF1, GAA, common gamma chain (CD132), adenosine deaminase, WAS protein, globin, alpha galactosidase A, delta-aminolevulinic acid (ALA) synthase, delta-aminolevulinic acid dehydratase (ALAD), hydroxymethylbilane (HMB) synthase, uroporphyrinogen (URO) synthase, uroporphyrinogen (URO) decarboxylase, coproporphyrinogen (COPRO) oxidase, protoporphyrinogen (PROTO) oxidase, ferrochelatase, alpha-L-iduronidase, idronate sulfatase, heparan sulfamidase, N-acetylglucosaminidase, heparan-alpha-glucosaminide, N-acetyltransferase, 3'-glucosidase ... It can encode N-acetylglucosamine 6-sulfatase, galactose-6-sulfatesulfatase, β-galactosidase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, and hyaluronidase.

[0441] In addition to the NOI, the vector may also contain or encode an siRNA, shRNA or regulatory shRNA (Dickins et al. (2005) Nature Genetics 37:1289-1295, Silva et al. (2005) Nature Genetics 37:1281-1288).

[0442] Indications The vectors of the present invention, including retroviral and AAV vectors, may be used to deliver one or more NOIs useful in the treatment of disorders listed in WO 1998 / 05635, WO 1998 / 07859, WO 1998 / 09985. The NOI of interest may be DNA or RNA. Examples of such diseases are described below.

[0443] Disorders responsive to: cytokine and cell proliferation / differentiation activity; immunosuppressant or immunostimulatory activity (e.g., for the treatment of immune deficiencies, including infection with the human immunodeficiency virus, for modulating lymphocyte proliferation, for the treatment of cancer and numerous autoimmune diseases, and for the prevention of graft rejection or the induction of tumor immunity); modulation of hematopoiesis (e.g., for the treatment of bone marrow or lymphatic system disorders); promotion of growth of bone, cartilage, tendon, ligament, and nerve tissue (e.g., for wound healing, burns, ulcers, and the treatment of periodontal disease and neurodegeneration); inhibition or activation of follicle-stimulating hormone (regulation of fertility); chemotactic / chemokinetic activity (e.g., after injury) or for recruiting specific cell types to sites of infection); hemostatic and thrombolytic activity (e.g., for the treatment of hemophilia and stroke); anti-inflammatory activity (e.g., for the treatment of septic shock or Crohn's disease); macrophage inhibitory and / or T cell inhibitory activity, and therefore, anti-inflammatory activity; anti-immune activity (i.e., inhibitory effects on cellular and / or humoral immune responses, including responses not related to inflammation); inhibition of the ability of macrophages and T cells to adhere to extracellular matrix components and fibronectin, and upregulated Fas receptor expression in T cells.

[0444] Malignant disorders, such as cancer, leukemia, benign and malignant tumor growth, invasion and spread, angiogenesis, metastasis, ascites and malignant pleural effusion.

[0445] Autoimmune diseases, such as arthritis, e.g. rheumatoid arthritis, hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other diseases.

[0446] Vascular diseases, such as arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome, cardiovascular effects, peripheral vascular disease, migraine and aspirin-dependent antithrombosis, stroke, cerebral ischemia, ischemic heart disease or other diseases.

[0447] Diseases of the digestive tract, such as peptic ulcer, ulcerative colitis, Crohn's disease and other diseases.

[0448] Liver disease, e.g., liver fibrosis, cirrhosis.

[0449] Inherited metabolic disorders, such as phenylketonuria (PKU), Wilson's disease, organic acidemias, urea cycle disorders, cholestasis and other diseases.

[0450] Kidney and urinary diseases, such as thyroiditis or other glandular diseases, glomerulonephritis or other diseases.

[0451] Disorders of the ear, nose and throat, for example, otitis or other ENT diseases, dermatitis or other skin diseases.

[0452] Dental and oral disorders, e.g., periodontal disease, periodontitis, gingivitis or other dental / oral diseases.

[0453] Testicular disease, e.g., orchitis or epididymo-orchitis, infertility, testicular trauma or other testicular disease.

[0454] Gynecological diseases, such as placental insufficiency, placental failure, habitual miscarriage, eclampsia, pre-eclampsia, endometriosis and other gynecological diseases.

[0455] Ophthalmological disorders, such as Leber's congenital amaurosis (LCA), e.g. LCA10, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, glaucoma, e.g. open-angle glaucoma and juvenile congenital glaucoma, intraocular inflammation, e.g. retinitis or cystoid macular edema, sympathetic ophthalmia, scleritis, retinitis pigmentosa, macular degeneration, e.g. age-related macular degeneration (AMD) and juvenile macular degeneration, e.g. Best's disease, Best's vitelliform macular degeneration, Stargardt's disease, Usher's syndrome, Doyne's honeycomb retinal dystrophy, Sorby's macular dystrophy trophy, juvenile retinoschisis, cone-rod dystrophy, corneal dystrophy, Fuchs' dystrophy, Leber's congenital amaurosis, Leber's hereditary optic neuropathy (LHON), Adie's syndrome, Oguchi's disease, degenerative fundus diseases, ocular trauma, ocular inflammation caused by infection, proliferative vitreoretinopathy, acute ischemic optic neuropathy, excessive scarring, such as after glaucoma filtration surgery, reactions to ocular implants, corneal transplant graft rejection, and other ophthalmic diseases, such as diabetic macular edema, retinal vein occlusion, RLBP1-related retinal dystrophy, choroideremia, and color vision defects.

[0456] Neurological and neurodegenerative disorders, e.g., Parkinson's disease, complications and / or side effects of treatment for Parkinson's disease, AIDS-related dementia syndrome, HIV-associated encephalopathy, Devic's disease, Sydenham's chorea, Alzheimer's disease and other degenerative diseases, CNS conditions or disorders, stroke, post-polio syndrome, psychiatric disorders, myelitis, encephalitis, subacute sclerosing panencephalitis, encephalomyelitis, acute neuropathies, subacute neuropathies, chronic neuropathies, Fabry's disease, Gaucher's disease, cystinosis, Pompe's disease, heterochromatin Leukodystrophies, Wiskott-Aldrich syndrome, adrenoleukodystrophy, beta thalassemia, sickle cell disease, Guillain-Barre syndrome, Sydenham chorea, myasthenia gravis, pseudotumor cerebri, Down's syndrome, Huntington's disease, CNS compression or CNS trauma or infection of the CNS, muscle atrophy and muscular dystrophies, diseases, conditions or disorders of the central and peripheral nervous system, motor neuron diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy, spinal cord and avulsion injuries.

[0457] other diseases and conditions, such as cystic fibrosis, mucopolysaccharidoses, e.g. Sanfilippo syndrome A, Sanfilippo syndrome B, Sanfilippo syndrome C, Sanfilippo syndrome D, Hunter syndrome, Hurler-Scheie syndrome, Morquio syndrome, ADA-SCID, X-linked SCID, X-linked chronic granulomatous disease, porphyria, hemophilia A, hemophilia B, post-traumatic inflammation, bleeding, coagulation and acute phase response, cachexia, anorexia, acute infections, septic shock, infections, diabetes, complications or side effects of surgery, complications and / or side effects of bone marrow transplantation or other transplants, complications and side effects of gene therapy, e.g. due to infection with a viral carrier or AIDS, for suppressing or inhibiting the humoral and / or cellular immune response for the prevention and / or treatment of graft rejection in the case of transplantation of natural or artificial cells, tissues and organs, e.g. cornea, bone marrow, organ, lens, pacemaker, natural or artificial skin tissue.

[0458] siRNA, microRNA and shRNA In certain other embodiments, the NOI comprises microRNA. MicroRNA is a very large group of small RNAs naturally produced in organisms, at least some of which regulate the expression of target genes. The original members of the microRNA family are let-7 and lin-4. The let-7 gene encodes a small, highly conserved RNA species that regulates the expression of endogenous protein-coding genes during helminth development. The active RNA species is initially transcribed as a precursor of about 70 nt, which is post-transcriptionally processed into a mature form of about 21 nt. Both let-7 and lin-4 are transcribed as hairpin RNA precursors, which are processed into their mature forms by the Dicer enzyme.

[0459] In addition to the NOI, the vector may contain or encode an siRNA, shRNA or regulatory shRNA (Dickins et al. (2005) Nature Genetics 37:1289-1295, Silva et al. (2005) Nature Genetics 37:1281-1288).

[0460] Posttranscriptional gene splicing (PTGS) triggered by double-stranded RNA (dsRNA) is a conserved cellular defense mechanism for controlling the expression of foreign genes. Random integration of elements such as viruses or transposons is thought to result in the expression of dsRNA that activates sequence-specific degradation of homologous mRNA or viral genomic RNA. This silencing process is known as RNA interference (RNAi) (Ralph et al. (2005) Nature Medicine 11:429-433). The RNAi mechanism involves processing long dsRNA into RNA duplexes of approximately 21 to 25 nucleotides (nt). These products are termed small interfering or silencing RNAs (siRNAs), which are sequence-specific mediators of mRNA degradation. In differentiated mammalian cells, dsRNA longer than 30 bp has been shown to activate the interferon response, leading to the inhibition of protein synthesis and nonspecific mRNA degradation (Stark et al., Annu Rev Biochem 67:227-64 (1998)). However, this response can be circumvented by using 21nt siRNA duplexes (Elbashir et al., EMBO J. Dec 3;20(23):6877-88 (2001), Hutvagner et al., Science. Aug 3,293(5531):834-8. Eupub Jul 12 (2001)), which allows gene function to be analyzed in cultured mammalian cells.

[0461] Pharmaceutical Composition The present disclosure provides pharmaceutical compositions comprising a lentiviral vector described herein, or a cell or tissue transduced with a viral vector described herein, together with a pharmaceutically acceptable carrier, diluent, or excipient.

[0462] The present disclosure provides a pharmaceutical composition for treating an individual by gene therapy, wherein the composition comprises a therapeutically effective amount of a lentiviral vector. The pharmaceutical composition can be for use in humans or animals.

[0463] The composition may include a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant. The selection of a pharmaceutical carrier, excipient, or diluent may be based on the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may include, in addition to, a carrier, excipient, or diluent, any suitable binder, lubricant, suspending agent, coating agent, solubilizer, and other carrier substances (e.g., lipid delivery systems) that may aid or enhance the entry of the vector into the target site.

[0464] Where appropriate, the compositions may be administered by one or more of the following means: by inhalation; in the form of a suppository or pessary; topically in the form of a lotion, solution, cream, ointment, or dusting powder; by use of a skin patch; or orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules, alone or mixed with excipients, or in the form of elixirs, solutions, or suspensions containing flavorings or coloring agents. Alternatively, they may be injected parenterally, for example, intracavernosally, intravenously, intramuscularly, intracranially, intraocularly, intraperitoneally, or subcutaneously. For parenteral administration, the compositions are best used in the form of a sterile aqueous solution, which may contain other substances, for example, sufficient salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration, the compositions may be administered in the form of tablets or lozenges, which may be formulated in the usual manner.

[0465] The lentiviral vectors described herein can also be used to transduce target cells or tissues ex vivo, and then introduce the target cells or tissues into patients in need thereof. An example of such cells can be autologous T cells. An example of such tissue can be donor cornea.

[0466] Variants, derivatives, analogs, homologs and fragments In addition to the specific proteins and nucleotides described herein, the present invention also encompasses the use of variants, derivatives, analogs, homologs and fragments thereof.

[0467] In the present context, a variant of any given sequence is a sequence in which a specific sequence of residues (including both amino acid and nucleic acid residues) has been modified so that the polypeptide or polynucleotide in question retains at least one of its intrinsic functions. Variant sequences can be obtained by addition, deletion, substitution, modification, substitution and / or mutation of at least one residue present in the naturally occurring protein.

[0468] The term "derivative" as used herein in reference to a protein or polypeptide of the invention includes any substitution, mutation, modification, replacement, deletion and / or addition of one (or more) amino acid residues from or to the sequence, provided that the resulting protein or polypeptide retains at least one of its endogenous functions.

[0469] The term "analog" as used herein with respect to a polypeptide or polynucleotide includes any mimetic, i.e., a compound that retains at least one of the endogenous functions of the polypeptide or polynucleotide that it mimics.

[0470] Typically, amino acid substitutions can be made, for example, from 1, 2, or 3 to 10 or 20 substitutions, provided that the modified sequence retains the desired activity or ability. Amino acid substitutions can include the use of non-naturally occurring analogues.

[0471] The proteins used in the present invention may also have deletions, insertions, or substitutions of amino acid residues, resulting in silent changes and resulting in functionally equivalent proteins. Intentional amino acid substitutions may be made based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues, as long as the intrinsic function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with uncharged polar head groups with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0472] Conservative substitutions may be made, for example, according to the following table: Amino acids in the same group in the second column and preferably in the same line in the third column may be substituted for each other. [Table 1]

[0473] The term "homolog" refers to an entity that has a certain homology to the wild-type amino acid sequence and the wild-type nucleotide sequence. The term "homology" can be equated with "identity."

[0474] In this context, a homologous sequence is considered to include an amino acid sequence that may be at least 50%, 55%, 65%, 75%, 85%, or 90% identical to the subject sequence, preferably at least 95%, 97%, or 99% identical. Typically, a homolog contains the same active site, etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e., amino acid residues having similar chemical properties / functions), in the context of the present invention, it is preferred to express homology in terms of sequence identity.

[0475] In this context, a homologous sequence is taken to include a nucleotide sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical to the subject sequence, preferably at least 95%, 97%, 98% or 99% identical. Although homology may also be considered in terms of similarity, in the context of the present invention it is preferred to express homology in terms of sequence identity.

[0476] Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percent homology or identity between two or more sequences.

[0477] The percent homology can be calculated over consecutive amino acids; that is, one sequence is aligned with the other and each amino acid in one sequence is directly compared to the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed over only a relatively short number of residues.

[0478] While this is a very simple and consistent method, it does not take into account, for example, the following case: if there is a single insertion or deletion in a nucleotide sequence, the insertion or deletion can cause the subsequent codon in an otherwise identical pair of sequences to be dropped from the alignment, potentially causing a large decrease in percent homology when a global alignment is performed. Therefore, most sequence comparison methods are designed to produce an optimal alignment that takes into account possible insertions and deletions without unduly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to attempt to maximize local homology.

[0479] However, more complex methods assign a "gap penalty" to each gap that occurs in the alignment, such that, given the same number of identical amino acids, sequence alignments with as few gaps as possible (which reflects a higher relatedness between the two compared sequences) receive a higher score than those with many gaps. An "affine gap cost" is typically used, which imposes a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. Higher gap penalties will, of course, result in optimized alignments with fewer gaps. Most alignment programs allow gap penalties to be modified. However, when using such software for sequence comparisons, it is preferable to use the default values. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0480] Therefore, calculating maximum percent homology first requires generating an optimal alignment, taking into account gap penalties. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Research 12:387). Examples of other software capable of performing sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. (1999) supra, Ch. 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410), and the GENEWORKS comparison tool suite. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al. (1999) supra, pp. 7-58 to 7-60). However, for some applications, it is preferable to use the GCG Bestfit program. Another tool called BLAST 2 Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol Lett (1999) 174(2):247-50; FEMS Microbiol Lett (1999) 177(1):187-8).

[0481] Although the final percent homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pairwise comparison. In practice, a scaled similarity score matrix is ​​commonly used that assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. One example of such a matrix commonly used is the BLOSUM62 matrix, the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use the public default values ​​or a custom symbol comparison table (if provided) (see the user manual for further details). For some applications, it is preferred to use the public default values ​​for the GCG package or the default matrix for other software, such as BLOSUM62.

[0482] Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

[0483] A "fragment" is also a variant, and this term typically refers to a selected region of a polypeptide or polynucleotide that is of functional interest or, for example, in an assay. Thus, a "fragment" refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

[0484] Such variants can be produced using standard recombinant DNA techniques, such as site-directed mutagenesis. Where an insertion is desired, synthetic DNA can be made that encodes the insertion, along with 5' and 3' flanking regions corresponding to the naturally occurring sequence on either side of the insertion site. The flanking regions contain convenient restriction sites corresponding to sites in the naturally occurring sequence, so that the sequence can be cleaved with appropriate enzymes and the synthetic DNA ligated into the cleavage. The DNA can then be expressed in accordance with the invention to give the encoded protein. These methods are merely illustrative of the many standard techniques known in the art for manipulating DNA sequences; other known techniques can also be used.

[0485] All variants, fragments or homologues of regulatory proteins suitable for use in the cells and / or modular constructs of the present invention retain the ability to bind to the cognate binding site of the NOI such that translation of the NOI is suppressed or prevented in viral vector production cells.

[0486] All mutant fragments or homologues of the binding site retain the ability to bind to the cognate RNA binding protein such that translation of the NOI is suppressed or prevented in viral vector producing cells.

[0487] Codon optimization Polynucleotides used in the present invention (including NOIs and / or components of vector production systems) may be codon-optimized. Codon optimization has been previously described in WO 1999 / 41397 and WO 2001 / 79518. Cellular usage of individual codons varies from cell to cell. This codon bias corresponds to a bias in the relative abundance of individual tRNAs in a cell type. By changing codons within a sequence such that they are tailored to match the relative abundance of the corresponding tRNA, expression can be enhanced. Similarly, expression can be reduced by deliberately selecting codons whose corresponding tRNAs are known to be rare in a particular cell type. In this way, an additional degree of translational control is available.

[0488] Many viruses, including retroviruses, utilize a large number of rare codons, and by changing these to match commonly used mammalian codons, enhanced expression of the gene of interest, e.g., the NOI or packaging components, in mammalian production cells can be achieved. Codon usage tables are known in the art for mammalian cells and a variety of other organisms.

[0489] Codon optimization of viral vector components has numerous other advantages. The modification of these sequences removes RNA instability sequences (INS) from the nucleotide sequences encoding the viral particle packaging components required for viral particle assembly in producer / packaging cells. At the same time, the amino acid sequences encoding the packaging components are retained, so that the viral components encoded by the sequences remain the same, or at least sufficiently similar so that the functionality of the packaging components is not impaired. Furthermore, in lentiviral vectors, codon optimization circumvents the need for Rev / RRE for export, making the optimized sequences Rev-independent. Codon optimization also reduces homologous recombination between various constructs within the vector system (e.g., between overlapping regions in the gag-pol and env open reading frames). Therefore, the overall effect of codon optimization is a significant increase in viral titer and improved safety.

[0490] In one embodiment, only the codons associated with the INS are codon-optimized, but in a much more preferred and practical embodiment, the sequences are codon-optimized in their entirety, with some exceptions, e.g., the sequence containing the gag-pol frameshift site (see below).

[0491] The gag-pol gene of lentiviral vectors contains two overlapping reading frames encoding gag-pol proteins. Expression of both proteins depends on a frameshift during translation. This frameshift occurs as a result of ribosomal "slippage" during translation. This slippage is thought to be caused, at least in part, by a ribosomal stalling RNA secondary structure. Such a secondary structure exists downstream of the frameshift site in the gag-pol gene. In the case of HIV, the overlapping region extends from nucleotide 1222 downstream of the gag start position (where nucleotide 1 is the A of the gag ATG) to the gag end position (nt 1503). Therefore, the 281-bp fragment spanning the frameshift site and the overlapping region of the two reading frames is preferably not codon-optimized. Retention of this fragment will allow for more efficient expression of the Gag-Pol protein. In the case of EIAV, the start of the overlap is considered to be at nt 1262 (where nucleotide 1 is the A of the gag ATG) and the end of the overlap is at bp 1461. To ensure that the frameshift site and gag-pol overlap are maintained, wild-type sequence is retained from nt 1156 to 1465.

[0492] Derivations from optimal codon usage can be made, for example, to accommodate convenient restriction sites, and conservative amino acid changes can be introduced into the Gag-Pol protein.

[0493] In one embodiment, codon optimization is based on a lightly expressed mammalian gene. The third base, and sometimes the second and third bases, may be modified.

[0494] It will be understood that due to the degeneracy of the genetic code, numerous gag-pol sequences are available to those skilled in the art. Also, numerous retroviral mutants have been described that can be used as a starting point for creating codon-optimized gag-pol sequences. Lentiviral genomes can be highly variable. For example, there are numerous quasi-species of HIV-1 that are still functional. This is also true for EIAV. These mutants can be used to enhance specific parts of the transduction process. Examples of HIV-1 mutants can be found in the HIV Databases operated by Los Alamos National Security, LLC at http: / / hiv-web.lanl.gov. Details of EIAV clones can be found in the National Center for Biotechnology Information (NCBI) database at http: / / www.ncbi.nlm.nih.gov.

[0495] The strategy for codon-optimizing gag-pol sequences can be used with any retrovirus. It will apply to all lentiviruses, including EIAV, FIV, BIV, CAEV, VMR, SIV, HIV-1, and HIV-2. This method could also be used to enhance expression of genes from HTLV-1, HTLV-2, HFV, HSRV, and human endogenous retroviruses (HERVs), MLV, and other retroviruses.

[0496] Codon optimization can render gag-pol expression Rev-independent. However, to enable the use of anti-rev or RRE elements in lentiviral vectors, it would be necessary to make the viral vector production system completely Rev / RRE-independent. Therefore, the genome would also need to be modified. This is achieved by optimizing the vector genome components. Advantageously, these modifications also result in the production of a safer system, free of all additional proteins in both producer and transduced cells.

[0497] In this example, it is demonstrated that the modified U1 of the invention and related methods and uses described herein can advantageously result in an acceptable or favorable safety profile.

[0498] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application, and nothing herein should be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0499] The present invention will now be described in more detail by way of examples, which are intended to serve to aid those skilled in the art in practicing the invention and are not intended to limit the scope of the invention in any way.

[0500] Example Common Molecular / Cell Biology Techniques and Assays Modified U1 snRNA expression construct DNA-based expression constructs for modified U1 snRNAs contain conserved sequences in endogenous U1 snRNA genes that drive RNA transcription and termination, highlighted below in the non-limiting example of 256U1 (also referred to as U1_256) snRNA. [ka]

[0501] A summary of the initial modified U1 snRNAs and controls used in this study is shown in the table below, which shows the novel annealing and target site sequences (sequences are displayed in the 5' to 3' direction).

[0502] Table I. Sequence listing showing target annealing sequences (heterologous sequences complementary to the target sequence) within the test modified and control U1 snRNAs and their target sequences used in the initial study. Nucleotides are shown as DNA because they are encoded within the respective expression cassettes in the "retargeting region." An (AT) motif was present in all initial constructs, which in each case constituted the first two nucleotides of the U1 snRNA molecule. Target sequence numbers, where indicated, refer to targets in the NL4-3 (GenBank: M19921.2) or HXB2 (GenBank: K03455.1) strains of HIV-1. This is because the lentiviral vector genomes in this study contained hybrid packaging signals composed of these two highly conserved strains (the packaging sequence used in this study is most similar to the vector sequence in GenBank: MH782475.1). [Table 2]

[0503] Adherent cell culture, transfection and lentiviral vector production HEK293T cells were maintained in complete medium [Dulbecco's modified Eagle's medium (DMEM) (Sigma) supplemented with 10% heat-inactivated (FBS) (Gibco), 2 mM L-glutamine (Sigma), and 1% non-essential amino acids (NEAA) (Sigma)] at 37°C in 5% CO .

[0504] Standard scale production of HIV-1 vectors in adherent form was in 10 cm dishes under the following conditions (if performed in other formats, all conditions were scaled by area): HEK293T cells were cultured at 3.5 x 10 in 10 mL of complete medium. 5cells / ml, and 24 h later, cells were transfected with the following mass ratio of plasmids per 10-cm plate: 4.5 μg genome, 1.4 μg Gag-Pol, 1.1 μg Rev, 0.7 μg VSV-G, and 0.01–2 μg modified U1 snRNA plasmid.

[0505] Transfections were performed by mixing DNA with Lipofectamine 2000CD in Opti-MEM according to the manufacturer's protocol (Life Technologies). After approximately 18 hours, sodium butyrate (Sigma) was added to a final concentration of 10 mM for approximately 5-6 hours, after which the transfection medium was replaced with fresh serum-free medium. Vector supernatants were typically harvested after 20-24 hours, then filtered (0.22 μm), and frozen at -20 / -80°C. As a positive control for nuclease treatment, Benzonase® was typically added to the harvest at 5 U / mL for 1 hour, followed by filtration.

[0506] Suspension cell culture, transfection and lentiviral vector production HEK293T.1-65s suspension cells were grown in a shaking incubator (25 mm orbit set at 190 RPM) in Freestyle + 0.1% CLC (Gibco) at 37°C in 5% CO2. All vector production using suspension was performed in 24-well plates (1 mL volume, on a shaking table), 25 mL shake flasks, or bioreactors ( < HEK293Ts cells were cultured at 8 × 10 per ml in serum-free medium (5 L). 5 Cells were seeded and incubated at 37°C in 5% CO with shaking throughout vector production. Approximately 24 hours after seeding, cells were transfected with the following mass ratio of plasmid per effective final volume of culture at the time of transfection: 0.95 μg / mL genome, 0.1 μg / mL Gag-Pol, 0.6 μg / mL Rev, 0.7 μg / mL VSV-G, and 0.01–0.2 μg / mL modified U1 snRNA plasmid.

[0507] Transfection was performed by mixing DNA with Lipofectamine 2000CD in Opti-MEM according to the manufacturer's protocol (Life Technologies). After approximately 18 hours, sodium butyrate (Sigma) was added to a final concentration of 10 mM. Vector supernatants were typically harvested after 20-24 hours, then filtered (0.22 μm) and frozen at -20 / -80°C. As a positive control for nuclease treatment, Benzonase® was typically added to the harvest at 5 U / mL for 1 hour, followed by filtration.

[0508] Lentiviral vector titration assay For titration of lentiviral vectors with GFP marker-containing cassettes, HEK293T cells were cultured in 96-well plates at 1.2 × 10 4 Cells were seeded at 1000 cells / well. A viral vector encoding GFP was used to transduce cells in complete medium containing 8 mg / ml polybrene and 1x penicillin-streptomycin for approximately 5-6 hours, after which fresh medium was added. Transduced cells were incubated at 37°C in 5% CO2 for 2 days. Cultures were then prepared for flow cytometry using an Attune-NxT (Thermofisher). GFP expression rates were measured, and 2x10 cells at the time of transduction were counted. 4 The vector titer was calculated using the expected cell number (based on typical growth rate), the dilution factor of the vector sample, the percentage of the positive GFP population, and the total volume at the time of transduction.

[0509] Lentiviral vector titration by integration assay For lentiviral vector titration by integration assay, 0.5 mL of vector supernatant from neat to 1:5 dilution was used to titer 1 x 10 vectors in a 12-well format in the presence of 8 μg / mL polybrene. 5 HEK293T cells were transduced. Cultures were passaged for 10 days (1:5 split every 2–3 days) and then 1 × 10 6Host DNA was extracted from the cell pellets. Duplex quantitative PCR was performed using FAM primers / probes targeting the HIV packaging signal (ψ) and RRP1, and vector titers (TU / mL) were calculated using the following factors: transduction volume, vector dilution, and RRP1-normalized HIV-ψ copy number (detected per reaction).

[0510] SDS-PAGE and immunoblotting Standard SDS-PAGE and immunoblotting were performed primarily on vector-terminated cells after vector recovery. For immunoblot analysis of vector particles, approximately 2 mL of filtered vector supernatant was centrifuged in a microfuge at 21,000 rpm at 4°C for 1-2 hours, and the "pellet" was then resuspended in 20-30 μL of PBS. These concentrated vector preparations were quantified by the PERT assay (described below), yielding 7 x 10 4 The PERT predicted TU vector was loaded per well of an SDS-PAGE gel. Approximately 1 × 10 6 Vector-terminated cells were lysed in 200 μl of fractionation buffer, and nuclei were removed by centrifugation. Protein samples were quantified by BioRad assay, and typically 5 μg of protein was loaded onto preformed 12-15 well 4-20% acrylamide gels. Proteins were transferred to nitrocellulose at 45 V for 3 hours on ice. Blots were blocked overnight at 4°C in 5% milk PBS / Tween-20. Blots were probed with primary and HRP secondary antibodies (typically diluted 1:100 in blocking buffer). Immunoblots were analyzed by ECL detection followed by X-ray film exposure.

[0511] RNA extraction and RT-qPCR assay Total RNA was extracted and purified from cell or LV samples using an RNeasy Mini Kit (QIAGEN). One microgram of RNA was treated with DNase I (Ambion) for 1 hour and then inactivated. 50 ng of DNase I-treated RNA was used in a qRT-PCR reaction consisting of Taqman® 1-Step RT-PCR Master Mix (Life Technologies) under standard chemical RT-PCR cycling conditions using a QuantStudio™ 6 (Life Technologies). Target-specific primer / probe sets were used. Negative control reactions contained no RT to control for DNA contamination.

[0512] Protein analysis by mass spectrometry Sample preparation Samples were prepared for MS analysis by in-solution trypsin digestion and subsequent peptide cleanup. Briefly, samples were denatured using a high-molarity urea buffer, followed by the addition of dithiothreitol (DTT) and iodoacetamide as reducing and alkylating reagents, respectively. After protein linearization and dilution to reduce the urea concentration, trypsin was added to each sample. Protein digestion was completed by overnight incubation at 37°C. Trypsin activity was then neutralized by adding trifluoroacetic acid (TFA) to an acidic pH. The digested peptides were then cleaned and desalted using a C18 column. The column was first activated with acetonitrile (ACN) and thoroughly washed with 0.1% TFA before loading the digested peptides. After further washing with 0.1% TFA, peptides were eluted with an acidified 70% ACN solution and collected in low-binding tubes. The ACN was then removed by evaporation using a SpeedVac, and the washed, desalted, dried peptides were resuspended in a solution containing 2% ACN and 0.1% formic acid (FA).

[0513] Liquid chromatography and mass spectrometry Peptides were analyzed on an UltiMate 3000 (Thermo Fisher Scientific) mass spectrometer connected online to a Q Exactive™ HF mass spectrometer (Thermo Fisher Scientific). Peptides were loaded onto a μPAC™ trapping column (PharmaFluidic, Ghent, Belgium) at a flow rate of 5 μL / min for 3 min and then separated on a 50 cm μPAC™ column (PharmaFluidics) using a 120 min nonlinear gradient of acetonitrile from 0.8% to 78% in 0.1% formic acid. The column temperature was maintained at 50 °C using the UltiMate 3000 column oven. The Q Exactive™ HF was operated data-independently (DIA) over the m / z range of 350 to 1,150. Full scan spectra were recorded at a resolution of 120,000 with an automatic gain control (AGC) target of 3 × 106, with a maximum injection time of 20 ms. After a full scan, 100 windows of 8.5 Th width with 0.5 Th overlap were used. DIA spectra were recorded at a resolution of 30,000 using an AGC target of 2 × 105 with a maximum injection time of 60 ms and a fixed initial mass of 200 Th. The normalized collision energy (NCE) was set to 28%, and the default charge state was set to 3. Peptides were ionized using an EASYspray electrospray emitter (Thermo Fisher Scientific) at a spray voltage of 2.0 kV and a heated capillary temperature of 250 °C.

[0514] Data analysis using DIA-NN Protein sequences from the Homo sapiens Uniprot reference proteome were concatenated with lentiviral proteins (GAG, POL) and frequently occurring contaminants to create the predicted spectral library for this project. Spectral libraries were predicted for all possible peptides with strict trypsin specificity (KR, not P) in the m / z range of 350–1,150, allowing for a maximum of one missing cleavage site. RT profiling was performed using an "any LC" quantification strategy, with a 5 ppm MS1 and MS2 profiling. 2 Mass spectra were analyzed in DIA-NN (version 1.7) using a fixed mass tolerance of 10 ppm for the spectra. The false discovery rate for precursor identification was set to 0.1%, and proteins were grouped according to their respective genes. Proteins were quantified using "normalized.unique" intensities.

[0515] Differential expression analysis Differential expression analysis was performed in R using the BioConductor DEP package. Proteins with missing values ​​were first filtered to ensure that at least one condition was quantified for all replicates. Variance-stabilizing normalization was performed using the BioConductor VSN package. Missing values ​​were then imputed using Quantile Regression Imputation of Left-Censored Data (QRILC). Differential expression analysis was performed using a protein-association linear model with empirical Bayes statistics. P values ​​were corrected for multiple testing using the Benjamini & Hochberg method.

[0516] Generation of CAR-T cells and evaluation of their functionality Cell transduction Peripheral blood mononuclear cells (PBMCs) from three healthy human donors were purchased from a commercial supplier. 1.5 × 10 per well were cultured in modified T cell medium containing 100 IU / mL of recombinant human IL-2 (12-well plates). 64.5 x 10 PBMCs 6 1.0 x 10 T cells per mL were cultured with 100 IU / mL CD3 / CD28 T cell expander beads. LV-CAR / LV-CAR[+256U1] were added to the relevant wells at an estimated part in infection (MOI) of 1.25 or 0.3, as indicated. 1.0 x 10 T cells per mL were cultured by increasing the volume of modified T cell medium containing 100 IU / mL recombinant human IL-2 (increasing the size of the culture vessel, if necessary). 5 Cells were maintained at a concentration of 1.0 × 10 viable cells per mL in cell freezing medium after 13 days in culture. 7 Cells were frozen at a concentration of viable cells. The percentage of transduced cells was determined by flow cytometry after 8 days of initial expansion and after 5 days of recovery.

[0517] Functionality Testing The cell lines used to test CAR-T cell responses were SKOV-3 (an ovarian cancer cell line that expresses high levels of 5T4l) and three acute myeloid leukemia (AML) cell lines: THP-1, Kasumi-1, and AML-193 (the latter a 5T4-negative cell line). Target cell lines were labeled with a fluorescent cell tracking dye to allow subsequent identification by flow cytometry. Approximately 1 x 10 5 1 x 10 CAR-T cells in a 96-well round-bottom plate 5 The cells were co-cultured in triplicate with each cell line. After 24 hours, an aliquot of culture supernatant was removed from each well for analysis of interferon gamma and granzyme B by cytometric bead array. After 40 hours, the cells were harvested and stained with fluorescent viability dyes. The percentage of nonviable target cells in each experimental well was determined by flow cytometry.

[0518] Example 1 To assess the effect of poly(A) signal mutants on transcriptional readthrough of the HIV-1 poly(A) site, we designed a GFP-poly(A) luciferase reporter cassette. The reporter encodes an upstream GFP ORF (to enable normalization of transfection efficiency), a standard 3'SIN-LTR sequence (including the RU5 sequence containing the HIV-1 poly(A) signal), followed by an IRES-Gluc sequence and an SV40 poly(A) signal. Thus, any readthrough of the HIV-1 poly(A) signal could be measured by luciferase activity normalized to GFP expression. The effects of two poly(A) signal mutations (pAM1 = AAUAAA > AACAAA; pAKO = deletion of AAUAAAA) and the wild-type poly(A) signal (wt pA = AAUAAA) were measured (Figure 2A).

[0519] Using a standard lentiviral vector genome and two genomes containing different 5' LTR poly(A) signal mutants (pAM1 or pAM2), vector particles were generated in the absence or presence of modified U1 snRNA (256_U1; supplied in parallel during production) and then titered. Vector produced in the presence of modified U1 snRNA was 3-6-fold higher than in the absence of modified U1 snRNA, independent of the presence or absence of a functional poly(A) site in the 5' LTR (Figure 2B). This indicates that modified U1 snRNA does not act to suppress leaky poly(A) readthrough (i.e., endogenous U1 snRNA binds to the major splice donor site and completely suppresses premature polyadenylation), and therefore must act to increase vector production through some other novel mechanism (perhaps by improving vRNA stability / nuclear export).

[0520] Example 2 Experiments were conducted to evaluate other features of the U1 snRNA molecule for their possible relevance in the present invention. Several modified U1 snRNA expression cassettes were constructed, all of which had target sequences for the "256" position in the HIV-1 packaging region (Figure 3A). Two mutants contained published mutations in the stem-loop I region that eliminated U1-70K protein binding (256_70K_m1 and 256_70K_m2), two mutants contained published mutations in the stem-loop II region that eliminated U1A protein binding (256_U1A_m1 and 256_U1A_m2), and one mutant contained a mutated Sm protein-binding motif (256_SM_m1) (Alexander, MR et al., 2010, Nucleic Acids Res., 38:3041-53; Ashe, MP et al., 2000, RNA, 6:170-7). Three other cassettes (U1A5, U1A6, and U1A7) expressing naturally occurring U1 snRNAs were also constructed. These contained the conserved Sm-binding region but had very different sequences within the cloverleaf structure (and were therefore unlikely to bind to U1-70K or U1A). Finally, two control U1 snRNA constructs targeting the lacZ gene sequence were generated as negative controls.

[0521] When these modified U1 snRNAs were individually cotransfected with lentiviral vector components (marker gene = GFP) into HEK293T producer cells, vector titers were enhanced 2-4 fold by 256_U1 and 70K or U1A protein-binding mutant U1, but not by other mutants (Figure 3A). U1A-based snRNAs increased LV titers independently of functional U1A-70K and U1A binding loops, whereas the titer increase depended on the Sm protein-binding motif. snRNA mutants U1A5, U1A6, and U1A7 did not increase LV titers. This indicates that some structural feature of the U1A snRNA is required for this effect.

[0522] Example 3 Experiments were conducted in the context of adhesive HEK293T vector production. Standard lentiviral vectors encoding GFP were produced in the absence or presence of modified U1 snRNA containing targeting sequences to sites along the length of the 5' end of the vector genome vRNA molecule, with targeting lengths of 15 or 9 nucleotides of complementarity. The modified U1 snRNAs are named according to the first nucleotide of the targeting sequence site along the length of the 5' end of the vector genome vRNA molecule (see Table I). The data show that an increased magnitude of vector titer enhancement correlates with targeting sites further away from the 5' polyA site, with the ideal target region being encoded within the SL1 loop of the packaging signal (Figure 4). The data also show that utilizing a targeting length of 15 nucleotides of complementarity instead of 9 nucleotides (as per the endogenous U1 snRNA) results in a more robust increase in vector titer.

[0523] Example 4 Experiments were performed in the context of suspension serum-free HEK293T vector production. Standard lentiviral vectors encoding GFP were produced in the absence or presence of modified U1 snRNA containing targeting sequences to sites along the length of the 5' end of the vector genome vRNA molecule, including a 15-nucleotide targeting length of complementarity. The modified U1 snRNAs are named according to the first nucleotide of the targeting sequence site along the length of the 5' end of the vector genome vRNA molecule. The data show that increasing magnitude of vector titer enhancement correlates with targeting sites further away from the 5' polyA site, with an ideal targeting region encoded within the SL1 loop of the packaging signal (Figure 5).

[0524] Example 5 Experiments were performed in the context of adherent HEK293T vector production. Standard lentiviral vector encoding GFP (pHIV-EF1a-GFP) or CD19 *A standard lentiviral vector (pHIV-EF1a-CD19) encoding a chimeric antigen receptor for HIV was produced in the absence or presence of modified U1 snRNA (256U1 or 305U1) targeting either site within the lentiviral vector packaging region or a modified U1 snRNA targeting a LacZ control (LacZU1). The modified U1 snRNAs are named according to the first nucleotide of the targeting sequence site along the length of the 5' end of the vector genome vRNA molecule (see Table I). The modified U1 snRNA expression construct was supplied at two different doses (i.e., 1x and 4x). The data demonstrate that the present invention can be applied independently of the vector genome payload (Figure 6).

[0525] *The CD19 CAR in this example and all other examples shown herein utilized an scFV based on publicly available sequence.

[0526] Heavy chain - GenBank CAA67618.1: QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTSVTVSS (SEQ ID NO: 191).

[0527] Light chain (kappa) - GenBank AAB34430.1: ELVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKRRS (SEQ ID NO: 192).

[0528] Example 6 - Promiscuous splicing from MSD, reduced titer of MSD-2KO lentiviral vector, and restoration / enhancement of titer by redirected U1 snRNA The general structure of lentiviral vector genomes is consistent across all third-generation vector systems (Toshie SAKUMA, Michael A. BARRY, and Yasuhiro IKEDA. Biochem. J. (2012) 443, 603-618), maintaining the position of the RRE upstream of the transgene cassette and the 5' region of the HIV-1 provirus containing the packaging sequence, but differing in other aspects, with later generations becoming U3 / tat independent, exhibiting self-inactivation in the 3' LTR, and including the use of cppt and wPRE. The apparent lack of examples of engineering the 5' packaging sequence is likely due to its complex structure, which encodes the condensed information required for multiple aspects of HIV-1 replication, transcription, balance in splicing, translation of GagPol, genome dimerization, assembly, reverse transcription, and integration. Within this complex region, the major splice donor (MSD) is embedded within the stem-loop 2 (SL2) region between SL1 (dimerization loop) and SL3 (binding to Gag). Relocation of the RRE sequence (and the associated splice acceptor 7 (sa7) in the envelope region) immediately downstream of the packaging region in the lentiviral vector genome was thought to "supply" the splice acceptor (sa7) to the MSD in the absence of rev. During lentiviral vector production, supply of rev was thought to result in binding of rev to the RRE and suppression of MSD splicing to sa7, resulting in the production of unspliced ​​full-length lentiviral vector genome vRNA (Figure 7A). However, we (Figure 9Bii) and others (e.g., Cui et al. (1999), J. Virol., 73:6171-6176) have shown that aberrant splicing from the MSD to splice acceptor sites within transgenic sequences can be substantial, resulting in relatively low levels (in some cases less than 5%) of unspliced ​​vRNA available for packaging into vector virions (compared to total; see Figure 7B).

[0529] Inefficiencies in the generation of vRNA for packaging are not always directly observed in the lentiviral vector titers obtained with transient transfection methods due to the large number of vector genome plasmids delivered to cells. Even when this type of aberrant splicing occurs, standard third-generation vector titers are typically above 1 x 10. 7 It is routinely possible to exceed 100 TU / mL. However, we anticipate that the problem of aberrant splicing is likely to become more significant when developing stable producer cell lines, where far fewer integrated vector genome cassettes may be present. Indeed, we typically find that genomic content is limiting in stable producer clones, and we hypothesize that MSD activity may contribute substantially to this limitation.

[0530] A perhaps less obvious consequence, namely (increased) expression of the transgene cassette during production, is observed in the generation of aberrantly spliced ​​mRNA from the MSD within the transgene cassette. Previously, the TRIP system has been developed to suppress transgene expression during lentiviral vector production (described in WO 2015 / 092440), allowing for the recovery of vector titers proportionally related to the adverse effect of a particular transgene protein on vector production. The inventors have found that efficient aberrant splicing (e.g., in standard lentiviral vectors containing an EF1a-driven cassette; see Figure 9) generates mRNA typically encoding the transgene. Without wishing to be bound by theory, in the case of an EF1a-driven cassette, the MSD splices to the strong EF1a splice acceptor, whereas in the case of other promoter-UTR sequences, the MSD "selects" for weaker potential splice acceptors, even in the presence of rev. The MSD appears to "turn a blind eye" to the RRE-sa7 sequence and prefer other sites more centrally in the vector genome, i.e., in the transgene promoter region. This may be a "residual" property of the HIV-1 5' packaging region because, in wild-type HIV-1, the MSD is typically spliced ​​to splice acceptors located in the middle and 3' end of the genome. Although the MSD can be aberrantly spliced ​​at multiple locations within the downstream vector sequence, only mRNAs that meet the criteria for nonsense-mediated decay (i.e., likely meet the criteria because they encode a protein [transgene protein]) are transported to (and / or are stable in) the cytoplasm, where they can then be translated. This places additional strain on the TRIP system to maintain repression of transgene-encoding mRNAs, resulting in less repressive control over a larger pool of mRNAs.Furthermore, the use of tissue-specific promoters (in part to avoid transgene expression during lentiviral vector production) can be "ineffective" by the cytoplasmic appearance of translatable mRNA encoding the transgene through this mechanism of aberrant splicing. Essentially, the transgene is expressed by a (typically strong) constitutive promoter that drives expression of the vector genome vRNA.

[0531] Thus, there are numerous reasons to generate MSD-mutated lentiviral vectors; indeed, other researchers have attempted to do so in U3 / tat-independent lentiviral vectors without success. We have found that mutations in the MSD in HIV-1 activate adjacent cryptic splice donor sites in SL2, resulting in significant levels of splicing. For this reason, we have used mutations in both the MSD and the nearby cryptic splice donor (crSD) (see Figure 15 ), and refer to this modification as "MSD-2KO" or "MSD2KO" or "MSD functional modification." Figure 9 shows that this double mutation is highly effective in eliminating aberrant splicing from the SL2 splicing region (including both the MSD and CrSD) to the potent EF1a splice acceptor during lentiviral vector production. As similarly reported by other researchers, MSD-2KO lentiviral vector genomes containing three different promoter-GFP transgene cassettes have also been shown to result in reduced vector titers (Figure 8 ). In Figure 9Bi, we also show that providing HIV-1 tat in trans can rescue the observed decrease in MSD-2KO lentiviral vector genome titer, although the amount of "aberrant" splice products (from the minor cryptic splice donor at SL4) increases (Figure 9Bii). Importantly, modified U1 snRNA redirected to a different region of the vector packaging signal has been shown to increase MSD-2KO lentiviral vector genome titer without increasing the presence of minor splice products (Figures 9 and 10).

[0532] Example 7 - Increased MSD-2KO lentiviral vector titer is not due to suppression of the 5' polyA site within the vector genome cassette To assess whether the present invention works to suppress the 5' polyA site, functional mutations were introduced into the 5' polyA site in MSD-2KO lentiviral vector genomes containing either an EF1a or CMV-driven GFP transgene cassette (Figure 11). The pAm1 polyA mutation is illustrated in Figure 2 (reproduced in Figure 11A for clarity) and demonstrates complete blockage of polyadenylation activity. Surprisingly, we found that mutation of the 5' polyA signal only partially increased the titer of the EF1a-GFP-containing MSD-2KO lentiviral vector genome and had virtually no effect on the CMV-GFP MSD2KO lentiviral vector genome, which appeared to be commensurate with the reducing effect of the MSD-2KO mutation (the MSD2KO mutation was less pronounced in the EF1a-containing genome). Importantly, in this experiment, supplying 305U1 molecules increased the titers of both standard lentiviral vector genomes (in which endogenous U1 snRNA could presumably completely suppress any residual 5' polyA activity) and MSD2KO / pAm1 lentiviral vector genomes (which had no possible 5' polyA activity), providing compelling evidence that the modified U1 snRNA supplied to restore titer of MSD mutant lentiviral vectors acts at a previously undescribed posttranscriptional step.

[0533] We next attempted to mutate the 70K and U1A-binding loops of 305U1 and 256U1 to assess whether this affected the observed increased titer of MSD2KO lentiviral vector genomes. Figure 12 shows that functional mutations in SL1 or SL2 within the modified U1 snRNA had no effect on the ability of these molecules to enhance MSD-2KO lentiviral vector titer when coexpressed during production; only the Sm protein-binding mutations blocked this activity. This demonstrates that the previously assumed essential 70K-binding property of redirected U1 snRNA in suppressing polyA activity is not critical in the present invention and provides further evidence that the modified U1 snRNA used to increase MSD mutant lentiviral vector titer functions via a novel mechanism.

[0534] To assess whether the titer increase provided by modified U1 snRNAs when applied to MSD-2KO lentiviral vectors differs in their "preferred" target site compared to standard lentiviral vectors (see Figures 4 and 5), we screened a panel of modified U1 snRNAs targeting different sites along the 5' region of the MSD-2KO lentiviral vector genome (see Table I) (Figure 14). This screen indicated that targeting to the packaging regions (SL1-3) is preferred, with a "hotspot" likely present within SL3. The screen was performed using modified U1 snRNAs with 15 nucleotides (or 9 nucleotides shown) of complementarity to the target site, since we previously demonstrated that titer increases can be more robust when complementarity lengths longer than 9 nucleotides are used with standard lentiviral vectors (see Figure 4). Indeed, we performed other experiments showing that for the MSD-2KO lentiviral vector, the increased potency observed with the modified U1 snRNA (targeting the "305" sequence) was observed with only 7 nucleotides of complementarity, but that in preferred use, using 10-15 nucleotides of complementarity is better due to the increased potency enhancement (Figure 13) and also because this minimizes possible "off-target" effects of the modified U1 snRNA.

[0535] Example 8 – Increased MSD-2KO lentiviral vector titer by modified U1 snRNA is independent of the type of splice donor mutation Figure 15A shows genetic modifications to the SL2 loop of the "MSD2KO" mutant of the MSD mutant lentiviral vector genome packaging region, which mutates both the MSD and a potential splice donor located downstream (the MSD2KO mutant is used in many of the non-limiting examples herein). To assess whether the effect of increased titer due to the use of modified U1 snRNA is in any way dependent on the specific modifications made to the MSD2KO mutant, we created three other splice donor region mutants: [1] "MSD2KOv2" also introduces two specific modifications within the MSD and potential donor sequences; [2] "MSD-2KOm5" replaces the entire SL2 loop with an artificial stem-loop; and [3] complete SL2 deletion thus removes the entire splice donor region (also called the splicing region). We then produced standard or MSD mutant lentiviral vector variants (containing an EFS-GFP expression cassette) in HEK293T cells in the presence or absence of modified U1 snRNA (256U1) and titered the vector supernatants (Figure 15B). Results showed that all four MSD mutant lentiviral vector variants were reduced compared to the standard vector, but all four could be enhanced by the use of modified U1 snRNA provided during lentiviral vector production. This indicates that there is no specific sequence dependency of splice donor region mutations by modified U1 snRNA. Interestingly, the MSD-2KOm5 variant was the least reduced and showed the greatest increase in production titer when produced in the presence of 256U1 molecules, regardless of the internal promoter used (comparing EFS, EF1a, CMV, and human PGK promoters).

[0536] Example 9 - Use of a modified U1 snRNA cassette encoded in cis within a lentiviral vector genome plasmid DNA backbone.

[0537] Previous examples herein have disclosed the use of modified U1 snRNA molecules during lentiviral vector production in trans by transient cotransfection of HEK293T cells with lentiviral vector component plasmids and a modified U1 snRNA-encoding plasmid. To assess whether the MSD mutant lentiviral vector genome cassette and the modified U1 snRNA cassette could be properly encoded within the same plasmid DNA molecule, three mutant constructs were cloned (Figure 16A). The MSD mutant lentiviral vector genome cassette (MSD-2KO mutant) was modified to insert the 256 U1 expression cassette in three different orientations relative to the lentiviral vector genome cassette and / or functional plasmid backbone sequence. These "cis" versions of the plasmid were used to produce MSD mutant lentiviral vectors in HEK293T cells and compared to the "trans" mode, in which the modified U1 snRNA plasmid was cotransfected with the unmodified MSD mutant lentiviral vector genome (Figure 16B). The results show that the titers of these "cis" forms of the plasmid were similar to the unmodified MSD mutant lentiviral vector genome in the presence of 256U1 supplied in the co-transfection.

[0538] Example 10 - Use of cell lines stably expressing modified U1 snRNA to enhance production of both standard or MSD-2KO lentiviral vectors The 305U1 expression cassette was stably integrated into HEK293T cells, and standard or MSD-2KO lentiviral vectors were produced by transient transfection with or without additional 305U1 plasmid DNA. Successful generation of stable cells demonstrated for the first time that modified U1 snRNA can be endogenously expressed in cells without cytotoxic effects. This indicates that modified U1 snRNA does not titer cellular factors involved in either the spliceosome or U1 snRNA synthesis, resulting in no off-target or off-target effects affecting normal cell viability. Lentiviral vector production titers indicated that the increased titer provided by modified U1 snRNA in both standard and MSD-2KO lentiviral vectors is possible with a stable supply of modified U1 snRNA (Figure 18). This will allow modified U1 snRNA to be easily integrated into lentiviral vector packaging and producer cell lines.

[0539] Example 11 - Further examples of the use of modified U1 snRNA to increase the titer of standard lentiviral vectors encoding therapeutic transgene cassettes Standard lentiviral vectors encoding either wild-type or codon-optimized human alpha1-antitrypsin (fused to a T2A-GFP reporter) or chimeric antigen receptor (for 5T4) driven by an EF1a promoter cassette were produced in serum-free suspension HEK293T cells in the presence or absence of 256U1. These vectors were titrated by integration assay or flow cytometry to assess GFP expression in target cells (Figure 17). These data show that 256U1 increased the titers of all vectors tested.

[0540] Example 12 - MSD mutant lentiviral vectors produce lower amounts of transgene protein during manufacturing Another benefit of eliminating aberrant splicing during lentiviral vector production is that it reduces the amount of transgene-encoding mRNA that leads to the production of the transgene protein. Transgene expression can have a significant impact on lentiviral vector production, leading to the development of the TRiP system to suppress transgene translation during viral vector production (described in WO 2015 / 092440). Briefly, the bacterial protein "TRAP" is co-expressed during vector production and binds to its "TRAP binding sequence" (tbs) inserted upstream of the transgene ORF in the 5'UTR, blocking scanning ribosomes.

[0541] During this study, we unexpectedly found that transgene-encoding mRNA can be efficiently produced from the "external" (CMV) promoter driving the vector genome cassette by splicing out from the SL2 major donor splice region to an internal splice acceptor site. The extent to which this occurs depends on the internal sequence between the cppt and the transgene ORF (i.e., the promoter-5'UTR sequence). Use of the EF1a promoter (containing a very strong splice acceptor) in the transgene cassette results in aberrant splicing from MSD in over 95% of all transcripts derived from the external promoter (see Figure 7). By comparing total GFP expression in production cultures of standard or MSD-2KO lentiviral vectors (Figure 19), we demonstrate that up to 80% of the transgene protein expressed during production is derived from aberrant splice products. We found that combining the MSD-2KO genotype with the TRiP system enhanced the reduction in transgene protein production.

[0542] Example 13 Co-expression of modified U1 snRNA targeted to the vRNA of lentiviral vectors results in increased vRNA in vector particle samples Experiments were performed in the context of adhesive HEK293T vector production. Standard lentiviral vectors encoding GFP (pHIV-EF1a-GFP) or a chimeric antigen receptor for CD19 (pHIV-EF1a-CD19) were produced in the absence or presence of modified U1 snRNAs (256U1 or 305U1) targeting either site within the lentiviral vector packaging region or a modified U1 snRNA targeting a LacZ control (LacZU1). The modified U1 snRNAs are named according to the first nucleotide of the targeting sequence site within the lentiviral vector packaging region (see Table I). The modified U1 snRNA expression constructs were supplied at two different doses (i.e., 1x and 4x). After treating the vector supernatant with DNase / RNase, total RNA was extracted from the vector particles, followed by RT-qPCR against the packaging region of the vRNA (Psi) to quantify the total vRNA copies present (Figure 20). The data show that co-expression of modified U1 sRNA resulted in an increase in vRNA within vector particles.

[0543] Example 14 Coexpression of modified U1 snRNA targeted to the vRNA of lentiviral vectors can lead to reduced transgene expression during vector production. Experiments were performed in the context of suspension serum-free adapted HEK293T vector production. During evaluation of the enhanced production of modified U1 snRNA from HIV-1-based lentiviral vectors encoding the α1-antitrypsin (α1AT) transgene (fused to GFP) (Figures 21 and 22), a relative increase in LV titer was observed for vectors pseudotyped with Sendai envelope (F / HN) (approximately 20-fold) compared with vectors pseudotyped with VSVG (approximately 3-fold). Vectors were produced in suspension HEK293T cells (and, where indicated, adherent HEK293T cells) and then titered on adherent HEK293T cells by both integration assays and flow cytometry. Post-transduction cell lysates were subjected to immunoblotting for transgene protein (and GFP) and β-actin, which demonstrated low but consistent suppression of transgene expression when 256U1 was provided in trans. Because Sendai F protein requires trypsin activation before transduction, this result indicates that the presence of α1AT in the harvest inhibited trypsin activation of the F protein. Thus, the apparent suppression of α1AT expression in p256U1-transfected cells resulted in a further enhancement of active vector titers. Without wishing to be bound by theory, this result is consistent with a mechanism of action by 256U1 in which vRNA is not only stabilized (presumably avoiding nuclear degradation) but can also be diverted from translation, which would otherwise result in the production of transgene protein. Indeed, it was recently reported that a pool of untranslated, full-length, unspliced ​​wild-type HIV-1 is actively packaged into virions (Chen et al. (2020), PNAS;117(11):6145-6155). In this particular case, these two surprising effects of 256U1 are additive, resulting in a 10-fold increase in vector production / activity.In the case of other therapeutic vector genomes where transgene expression may be detrimental to output vector titer, this modest effect of reduced transgene expression by modified U1 snRNA may have a similar effect contributing to increased titer.

[0544] Example 15 Use of modified U1 snRNA to increase the titer of lentiviral vectors containing inverted transgene cassettes.

[0545] In some cases, it is necessary to utilize lentiviral vector genomes in which the transgene cassette is inverted, i.e., the transcription unit is opposite to the promoter driving the vector genome cassette. For example, most lentiviral vectors developed for the treatment of sickle cell disease or β-thalassemia contain the β-globin gene, in which the intron is encoded in a three-exon context (because out-splicing of the intron in terminally differentiated erythrocytes is required for efficient β-globin expression). In most cases, the intron can be retained in packaged lentiviral vector vRNAs containing the rev response element (RRE), because binding of rev to the RRE in the nucleus results in export of the intron-containing vRNA. However, this is not the case for the β-globin gene; if the transgene cassette is in the "forward" orientation, the intron is lost from these vRNAs even in the presence of rev / RRE. Additionally, cases can exist in which one component of the transgene cassette is in the reverse orientation and another component is in the forward orientation. For example, when a bidirectional transgene cassette or multiple separate cassettes are used.

[0546] To assess whether the use of modified U1 snRNAs allows for the increase of lentiviral vectors containing inverted transgene cassettes, lentiviral vectors containing the β-globin gene were produced in suspension (serum-free) HEK293T cells in the absence or presence of four different modified U1 snRNAs targeted to the packaging region (Figure 23). Experiments were performed in the context of suspension serum-free adapted HEK293T vector production. The data indicate that modified U1 snRNAs can also increase the titer of these classes of lentiviral vectors.

[0547] Example 16 The increased lentiviral vector titer with modified U1 snRNA is measurable in concentrated vector preparations. Experiments were performed in the context of suspension serum-free adapted HEK293T vector production. Lentiviral vectors encoding a firefly luciferase-GFP dual reporter transgene cassette were produced in suspension (serum-free) HEK293T cells in the absence or presence of 256U1. The clarified vector harvest was concentrated by centrifugation (approximately 20-fold concentration factor), and both pre- and post-concentration vector samples were then titered by transduction of adherent HEK293T cells followed by flow cytometry (Figure 24). The data show that the increased titer conferred by modified U1 snRNA is observed in processed LV preparations, providing further evidence that the enhanced vector titer is not an artifact associated with crude vector material.

[0548] Example 17 We developed a highly sensitive quantitative detection method for modified U1 snRNA to measure residual modified U1 snRNA in cell lysates and vector material after production. After pre-snRNA is processed in the cytoplasm, it is re-transported into the nucleus as part of the spliceosome, a central complex involved in pre-mRNA splicing. Therefore, the modified U1 snRNA described herein is expected to be primarily located in the nucleus, making it highly unlikely to be incorporated into virions. Indeed, other researchers have shown that processed U1 snRNA is not actively packaged into HIV-1 virions, and the presence of pre-U1 snRNA is 100-fold lower than the most abundant cellular RNAs (e.g., 7SL) detected in virions (Eckwahl et al. (2016); RNA, 22(8):1228-1238).

[0549] Nevertheless, to enable evaluation of intracellular expression of modified U1 snRNA and detection / quantification of residual RNA derived from modified U1 snRNA in vector products, we developed a TaqMan-based RT-qPCR assay (Figure 25). To distinguish modified U1 snRNA from highly expressed endogenous U1 snRNA, an amplicon was designed in which the forward primer was homologous to the vRNA-targeting sequence at the 5' end of the modified U1 snRNA molecule (see Figure 25A). Thus, while the reverse primer enabled cDNA synthesis of both endogenous and modified U1 snRNA, the quantitative PCR step occurred only from cDNA derived from modified U1 snRNA. In this non-limiting example, an 88-bp amplicon was designed to amplify 256 U1 snRNA. To evaluate the sensitivity of the primer / probe set, total RNA was extracted and purified from replicate cultures of suspension (serum-free) HEK293T cells transfected with p256U1 and treated with benzonase (to degrade residual p256U1 DNA). To assess the signal derived from undigested p256U1 DNA, Taqman qPCR was performed on purified total RNA with or without a reverse transcriptase step (see Figure 25B). The p256U1 plasmid was used as a standard curve for the qPCR step, demonstrating excellent linearity and range. Diluted cDNA samples from untransfected and p256U1-transfected cellular RNA (+RT treatment) showed Ct values ​​that differed by 19–20 cycles, with the RT-untreated p256U1-transfected cellular RNA exhibiting only approximately two-fold lower Ct values ​​than the untransfected cellular RNA. This demonstrated that the RT-qPCR assay could clearly detect and quantify modified U1 snRNA rather than endogenous U1 snRNA.

[0550] Example 18 Modified U1 snRNA dose-response and polydispersity modeling to optimize the ratio of modified U1 snRNA plasmid to LV component plasmid for maximal transient transfection of suspension (serum-free) HEK293T cells A simple dose-response study was performed to assess the relationship between the input modified U1 snRNA plasmid during transient transfection, resulting modified U1 snRNA expression, and the effect on both vector genomic RNA (vRNA) and output vector titer. LVs encoding an EF1a promoter-driven CAR-CD19 expression cassette were produced in suspension (serum-free) HEK293T cells by transient transfection of LV component pDNA. In this case, the input of p256U1 was set over a range of amounts from 0 to 600 ng / mL (effective final culture concentration at transfection). The ratio / amount of all LV pDNA remained constant, while total pDNA was maintained by filling with pBlueScript. To normalize total RNA within the cDNA step, cells were analyzed post-production by extracting total RNA and quantifying both 256U1 snRNA and vRNA, as well as an endogenous transcript (RPH1; data not shown) (Figure 26A). Additionally, clarified LV supernatants were titered by integration assay (Figure 26B). The data show a linear correlation between the amount of input p256U1, the expression level of 256U1 snRNA, and the steady-state level of vRNA (which increased linearly). The production titer of LV-CARCD19 also showed a linear relationship with the amount of input p256U1 (and the increase in vRNA in producer cells) up to an input level of 300 ng / mL, where the titer increase was greatest. This suggests that the minimum input level of p256U1 to achieve the maximum titer increase (under these conditions and for this LV genome) is somewhere between 150 and 300 ng / mL.

[0551] Using design of experiments (DoE), 28 conditions were established and tested at the 40 mL shake flask scale. In this case, p256U1, pGenome (in this case, pHIV-EF1a-5T4CAR), and pVSVG were varied while maintaining constant pGagPol / pRev input levels (Figure 27). The clarified vector harvest was titrated by transduction of adherent HEK293T cells followed by immunoflow cytometry using MAbs against 5T4CAR. The optimal input of 180 ng / mL p256U1 identified by DoE was then applied to the production of other HIV-EF1a-5T4CAR vector preparations, resulting in an average increase in LV production titers of approximately 10-fold compared to the absence of p256U1. The 180 ng / mL input of p256U1 closely matched the findings of dose-response experiments (see Figure 26).

[0552] Example 19 Case Study: Generation and Evaluation of CAR-T Cells Using Enriched / Purified Lentiviral Vector Preparations Produced in the Presence or Absence of 256U1. A small-scale case study was conducted to evaluate the impact of applying modified U1 snRNA to a lentiviral vector gene therapy product. In this case, a lentiviral vector encoding a chimeric antigen receptor targeted to 5T4 (HIV-EF1a-5TACAR / 'LV-CAR') was [1] produced and purified in the presence or absence of 256U1 (Figure 28), [2] the amount of residual 256U1 snRNA in the enriched product was quantified (Figure 29), [3] comparative protein analysis of the two enriched vector preparations was performed (Figure 30 and Table IV), [4] primary T cells expanded from three healthy donor PBMCs were transduced and their killing activity against 5T4-positive cells was assessed (Figures 31-33), and [5] residual 256U1 and vRNA in CAR-T expansion cultures were assessed by RT-qPCR (Figure 34).

[0553] Two concentrated HIV-EF1a-5TACAR vector preparations were generated in 250 mL shake flask-scale suspension (serum-free) HEK293T cell cultures in the presence of added p256U1 plasmid DNA (+256U1) or pBluescript as a negative control. The clarified harvest (not treated with benzonase) was then subjected to ion exchange chromatography to a concentration of approximately 125x, followed by nuclease treatment. Finally, the vector preparation was centrifuged for approximately 45 minutes and then resuspended in TSSM to a total concentration of 250x. Both the clarified harvest and concentrated vector samples were titrated by transduction of adherent HEK293T cells followed by immunoflow cytometry using an antibody against the CAR transgene (Figure 28). These data demonstrate the increased titer resulting from the use of 256U1 snRNA.

[0554] Vector samples from the enrichment process were then subjected to total RNA extraction. In this case, clarified harvest samples were treated with or without benzonase prior to extraction. Both vRNA and residual 256U1 snRNA were then quantified by RT-qPCR (see Example 17 for 256U1 RT-qPCR assay development). Figure 29 presents this data, showing: [1] the increase in vRNA in all vector samples from vectors produced in the presence of 256U1; [2] the effect of nuclease treatment on vRNA abundance relative to 256U1 snRNA; and [3] the relative ratio of 256U1 snRNA signal relative to vRNA in enriched vectors. This analysis indicates that the 256U1 snRNA signal present in the clarified LV harvest is primarily "free" RNA, likely derived from leaky / ruptured cells during production, since benzonase treatment dramatically reduces its detection. In concentrated LV material, the ratio of 256U1 to vRNA signal was 1 to 32, indicating the presence of a single 256U1 snRNA (or at least an 88-bp amplicon) for every 16 vRNA-containing LV particles. This suggested that 256U1 snRNA is not actively packaged within LV particles and that the signal detected in concentrated LV material was likely due to the presence of residual 256U1 snRNA outside the particles. This also suggests that further reduction of the 256U1 snRNA signal is possible through optimization of nuclease treatment and LV purification steps.

[0555] The two concentrated LV-CAR vector preparations were then analyzed by mass spectrometry to assess the primary effect of 256U1 snRNA expression during LV-CAR production on the protein mark-up of virions. Samples were prepared as described herein (see " Common Molecular / Cell Biology Techniques and AssaysPeptides were analyzed on an UltiMate 3000 connected online to a Q Exactive™ HF mass spectrometer (see the "" section). Mass spectra were analyzed in DIA-NN (version 1.7). Protein sequences from the homo sapiens uniprot reference proteome were concatenated with lentiviral proteins (gag, pol, rev) and VSV-G glycoproteins and frequent contaminants to create a predicted spectral library for this project. The top 400 proteins detected in the LV-CAR[+256U1] vector sample were ranked, and their relative abundances were plotted as a percentage of the total spectral abundance of the top 400 proteins (Figure 17, filled circles). The relative abundances of the same protein hits within the LV-CAR vector sample were also plotted to assess whether significant differences in the protein profiles of the two vector preparations could be visualized (Figure 17, open circles). These data demonstrate the presence of the predicted lentiviral proteins gag (rank #1), VSV-G (rank #2), and pol (rank #37), with rev appearing at rank #400. Comparison of the gag to pol peptide ratios showed a very similar ratio of approximately 16:1 for both samples. This is close to the 20:1 ratio for the allowed differential translation of gag / pol mRNA (as in wild-type HIV-1) (the gag polyprotein is the major product of translation, but approximately 1 in 20 frameshift events lead to translation of the gag pol polyprotein). Also present were the following cellular proteins known to be incorporated into HIV-1-based lentiviral virions: basigin (rank #3), HSPc-71K (rank #5), and cyclophilin A (rank #22; specifically binds to the capsid). Analysis revealed minimal differences between the two LV-CAR vectors. This indicates that overexpression of 256U1 snRNA during LV production did not result in a global up / downregulation of cellular proteins and / or their incorporation into LV virions.

[0556] Differential expression analysis of hits that showed significant differences of up to two-fold between the two LV samples was performed in R using the BioConductor DEP package. A selection of the key hits from this analysis is shown in Table IV. This statistical analysis showed that the two-fold increase in gag, pol, VSVG, and cyclophilin A was highly significant, suggesting that these LV proteins were more abundant in the LV-CAR[+256U1] vector compared to other background (potentially contaminating) proteins.

[0557] Table IV: Protein selection from statistical analysis performed on hits with a maximum 2-fold variation from comparative mass spectrometry protein analysis of enriched / purified preparations of HIV-EF1a-5T4CAR (LV-CAR) produced in the presence or absence of 256U1. Differential expression analysis was performed in R using the BioConductor DEP package. Proteins with missing values ​​were first filtered to ensure that at least one condition was quantified for all replicates. Variance-stabilizing normalization was performed using the BioConductor VSN package. Missing values ​​were then imputed using Quantile Regression Imputation of Left-Censored Data (QRILC). Differential expression analysis was performed using a protein-association linear model with empirical Bayesian statistics. P values ​​were corrected for multiple testing using the Benjamin & Hochberg method. Ranking numbers represent the top 400 proteins detected in the LV-CAR[+256U1] sample. [Table 3]

[0558] Two concentrated HIV-EF1a-5TACAR / LV-CAR vector preparations were used to transduce peripheral blood mononuclear cells (PBMCs) from three healthy donors at an MOI of 1.25 (both vector preparations) or an MOI of 0.3 (HIV-EF1a-5TACAR +256U1 only), followed by expansion of transduced T cells and cell banking on day 13. Cells were then recovered and expanded for an additional 5–6 days. Total viable cells and transduction rates with LV-CAR or LV-CAR[+256U1] vectors were monitored during expansion and after recovery (Figure 31). This analysis showed that despite using a matching MOI of 1.25 for both LV preparations, the LV-CAR[+256U1] vector generally transduced expanded T cells more efficiently than the LV-CAR vector (presumably due to fewer contaminants in the LV-CAR[+256U1] vector; see Figure 30 and Table IV). The transduction rate with the LV-CAR[+256U1] vector at an MOI of 0.3 was similar to that with the LV-CAR vector (MOI of 1.25), and therefore further comparisons were focused on these samples.

[0559] T cells expressing the 5T4CAR were then evaluated for target cell killing activity by co-incubation with equal numbers of 5T4-positive (THP-1, Kasumi-1, SKOV-3) or 5T4-negative (AML-193) cells, followed by analysis of cytokine release 24 hours after incubation (Figure 32) and cell killing 40 hours after incubation (Figure 33). These results indicate that CAR-T cells transduced with either of the two vector preparations were equally capable of being specifically activated in the presence of 5T4-positive cells by releasing granzyme B and interferon-γ, resulting in specific cell killing.

[0560] On days 8 and 13 posttransduction, cell samples were harvested and total RNA was extracted to assess the relative abundance of residual 256U1 snRNA compared to RPH1 mRNA, which was used as a cellular transcript loading control. Figure 34 shows these data, which indicate that the residual 256U1 snRNA signal was 4-5 logs lower and the residual vRNA was 3-3.5 logs lower compared to the RPH1 transcript at each time point. The decrease in the residual 256U1 snRNA signal between days 8 and 13 was approximately 5-fold greater than that of vRNA. This indicates that the majority of the residual 256U1 snRNA was exposed (for degradation) compared to vRNA (which is likely protected within the capsid), likely reflecting the presence of only a small number of intact LV virions that remained bound to T cells during proliferation. Overall, residual RNA analysis indicates that modified U1 snRNA can be detected at low levels in the processed LV material (1 in every 16 complete virions at this laboratory scale), but that at least 80% of this signal is likely present outside the virion, implying that further reduction of this residue may be possible through optimization of nuclease treatment and purification steps.

[0561] Given that constitutive, long-term expression of modified U1 snRNA is possible in the HEK293T cell line without apparent general cytotoxicity (see Example 21), delivery of full-length modified U1 snRNA to a fraction of target cells during LV transduction is unlikely to affect target cell viability. The design of the modified U1 snRNA would preclude specific interactions with host cell RNA, and its relative abundance in target cells compared with the vast pool of endogenous U1 snRNA would mean that it is highly unlikely to be able to efficiently compete with RNP factors for binding.

[0562] Example 20 Use of modified U1 snRNA to increase LV production from suspension (serum-free) packaging cells. The HIV-EF1a-CAR-CD19 vector or a GFP reporter mutant vector (HIV-EF1a-CAR-CD19-T2A-GFP) was produced in a lentiviral vector packaging cell line (PAC) in either shake flasks or 250 mL bioreactors (AMBR250) in the presence or absence of p256U1. HEK293T cells were transfected with all vector components in parallel as a control. Clarified vector harvests were titered by transduction of adherent HEK293T cells and subsequent integration assays. Titers were plotted against "without 256U1" (Figure 35). The data show that the increase in vector titer was greatest in the PAC cell line compared to HEK293T cells.

[0563] Example 21 Increased lentiviral vector titer in suspension (serum-free) HEK293T cells stably transfected with a 256U1 expression cassette. Suspension (serum-free) HEK293T cells were stably transfected with a 256U1-HygR expression cassette, and clones were selected. Clone 256U1c39 was selected for evaluation. Increased lentiviral vector titer was assessed at weeks 1, 5, and 10 after isolation and compared to parental HEK293T cells at each time point by continuously growing the cells in the presence or absence of hygromycin B and producing the HIV-EF1a-5T4CAR vector by transient transfection in the presence or absence of p256U1 (Figure 36). The data show that 256U1 snRNA expression in the 256U1c39 clone was stable in the presence or absence of hygromycin selection, indicating that long-term expression of these modified U1 snRNAs is not toxic to HEK293T cells. Additionally, the level of vector titer increase in the 256U1c39 cell line was close to the maximum titer increase observed across all conditions.

[0564] Example 22 The effect of increasing modified U1 snRNA on lentiviral vectors does not appear to be dependent on the conserved 5' dinucleotide "AU" present in endogenous U1 snRNA.

[0565] In Example 1, it is shown that the mechanism of action of the modified U1 snRNA is not due to polyA suppression of the 5' polyA signal within the 5' LTR region of the lentiviral vector (a known property of endogenous U1 snRNA), because the modified U1 snRNA is still able to increase the titer of LV containing a functional mutation of this polyA site.

[0566] Other researchers have characterized an aspect of U1 snRNA biology that appears to be important for its role in splicing (Yeh et al. (2017) Nucleic Acids Res. 45(16):9679-9693). Endogenous U1 snRNA recruits the CAP-binding complex (CBC) to its 5' end, which is important for U1 snRNP function in splicing. The authors found that the "AU" dinucleotide provides optimal binding of the CBC compared to other dinucleotides, highlighting why the "AU" dinucleotide is so widely conserved within eukaryotes.

[0567] To assess the importance of the conserved "AU" dinucleotide sequence in the context of modified U1 snRNA targeted to the packaging signal of lentiviral vector genomes, a number of mutants based on the 256 U1 snRNA were generated (Table V).

[0568] Table V: Mutant modified U1 snRNAs targeting position 256 of the HIV-1 LV vRNA genome were generated to assess the effect of changing the 5'-terminal dinucleotide on vector titer increase. This table shows how the 15-nucleotide variant 256U1 target sequence and base pairs in the vRNA compare to the 13-nucleotide variant target sequence. The 256U1_13 variant was designed to maintain 13 consecutive base pairs to the target where possible, resulting in a calculated T-melting temperature (Tm°C) of approximately 46°C. The dinucleotides at the 5' end of the modified U1 snRNA molecule are shown, and the underlined nucleotides represent possible transcription start sites based on the findings of Yeh et al. (2017). Variants indicated with an asterisk indicate that the first of the two indicated dinucleotides is likely not the first nucleotide of the modified snRNA. The transcription start site of the U1 promoter is also shown, and in the case of variants with "aa" and "cc," a likely TSS (1) is indicated (gray boxed "C"). [Table 4] TIFF0007787064000014.tif56151

[0569] Yeh et al. (2017) also reported on the effects of altering the first one or two nucleotides on transcription start site and U1 snRNA abundance. They found that transcription initiation was preferred at purines over pyrimidines; therefore, when pyrimidine-purine or pyrimidine-pyrimidine dinucleotides were placed at positions 1 and 2, the pyrimidine was "skipped" in favor of the next purine. Exceptions to this general rule were "UU" (in which case transcription initiation occurred 19 or 29 nucleotides downstream) or "AA" / "CC" (in which transcription initiation occurred at position -1 (C)). The "UU" variant was not present in the panel of 256 U1 variants tested. In the variant panel, the length of the target sequence was reduced from 15 to 13 nucleotides to allow testing of a large number of different dinucleotide variants in which one, both, or neither nucleotides could be involved in base pairing with the target sequence, depending on the predicted transcription start site (Yeh et al. (2017)). It was shown that modified U1 snRNAs containing targeting sequences of 9 to 15 nucleotides could all result in increased potency (Figure 37).

[0570] Therefore, the total length of the flanking targeting sequence was 13 nucleotides for all variants (although not exactly the same 13 nucleotides), and the T melting temperature for all mutants was predicted to be approximately 46°C. The HIV-EF1a-GFP vector was produced at a 24-well scale in suspension (serum-free) HEK293T cells, and each of the dinucleotide mutant U1 snRNAs was individually cotransfected with the vector components. The clarified vector harvest was titrated by transduction of adherent HEK293T cells followed by flow cytometry. The data shown in Figure 38 show the relative vector titers compared to "no 256U1." This indicates that all dinucleotide mutants except 256_13_Gt were able to increase vector titers, with most achieving a similar magnitude of increase as the control 256_13_aT U1 snRNA. Furthermore, there did not appear to be a correlation between the predicted CBC binding score of each dinucleotide mutant (according to Yeh et al. (2017)) and each mutant's ability to confer increased vector titers. This indicates that the known CAP-binding property of U1 snRNA in generating a competent U1 snRNP splicing complex is not critical for the vector titer-enhancing effect of the modified U1 snRNA described herein. Considering that mutant 256_13_gT (which has the same dinucleotide as 256_13_Gt) was able to confer increased vector titer, this also indicates that there may not necessarily be a dinucleotide to avoid (other than the aforementioned "UU")...

Claims

1. A modified U1 snRNA that has been modified to bind to a nucleotide sequence within a packaging region of a lentiviral vector genome sequence, wherein the packaging region of the lentiviral vector genome sequence is from the start of the 5'U5 domain to the end of a gag gene-derived sequence, and the modified U1 snRNA has been modified to introduce a heterologous sequence that is complementary to the nucleotide sequence.

2. 2. The modified U1 snRNA of claim 1, wherein the modified U1 snRNA is modified at the 5' end to introduce the heterologous sequence within 9 nucleotides from positions 3 to 11.

3. The modified U1 snRNA of any one of claims 1 to 2, wherein the modified U1 snRNA is modified at the 5' end to introduce the heterologous sequence within a natural splice donor annealing sequence.

4. 4. The modified U1 snRNA of claim 3, wherein 1 to 9 nucleic acids of said native splice donor annealing sequence are replaced by said heterologous sequence.

5. The modified U1 snRNA of any one of claims 1 to 4, wherein the modified U1 snRNA is modified at the 5' end to replace a sequence comprising a natural splice donor annealing sequence with the heterologous sequence complementary to the nucleotide sequence.

6. The modified U1 snRNA of any one of claims 1 to 5, wherein the heterologous sequence comprises at least 9 nucleotides complementary to the nucleotide sequence.

7. The modified U1 snRNA of any one of claims 1 to 6, wherein the heterologous sequence comprises 15 nucleotides complementary to the nucleotide sequence.

8. The modified U1 snRNA of any one of claims 1 to 7, wherein the nucleotide sequence is located within the 5'U5 domain, the PBS element, the SL1 element, the SL2 element, the SL3ψ element, the SL4 element, and / or a sequence derived from the gag gene.

9. The modified U1 snRNA of any one of claims 1 to 8, wherein the nucleotide sequence is located within the SL1, SL2 and / or SL3ψ element.

10. The modified U1 snRNA according to any one of claims 1 to 9, wherein said nucleotide sequence is located within the SL1 and / or SL2 element.

11. The modified U1 snRNA of any one of claims 1 to 10, wherein the nucleotide sequence is located within the SL1 element.

12. The modified U1 snRNA of any one of claims 1 to 11, wherein the modified U1 snRNA is a modified U1A snRNA or a modified U1A snRNA variant.

13. The modified U1 snRNA according to any one of claims 1 to 12, wherein the first two nucleotides at the 5' end of the modified U1 snRNA are not AU.

14. The modified U1 snRNA of any one of claims 1 to 13, wherein the lentiviral vector is derived from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or Visna lentivirus.

15. The modified U1 snRNA of any one of claims 1 to 14, wherein the lentiviral vector is derived from HIV-1, HIV-2 or EIAV.

16. The modified U1 snRNA of any one of claims 1 to 15, wherein the lentiviral vector is derived from SIV.

17. An expression cassette comprising a nucleotide sequence encoding the modified U1 snRNA according to any one of claims 1 to 16.

18. A cell for producing a lentiviral vector comprising a nucleotide sequence encoding vector components including gag, env, rev and the RNA genome of a lentiviral vector, and at least one nucleotide sequence encoding a modified U1 snRNA according to any one of claims 1 to 16.

19. A cell comprising the modified U1 snRNA of any one of claims 1 to 16.

20. 20. The cell of claim 19, wherein the cell further comprises a nucleotide sequence encoding the RNA genome of a lentiviral vector.

21. 21. The cell of claim 19 or claim 20, wherein the cell further comprises a nucleotide sequence encoding a nucleotide of interest.

22. 22. The cell of claim 21, wherein the nucleotide of interest provides a therapeutic effect.

23. 23. The cell of claim 22, wherein the nucleotide of interest encodes an enzyme, a cofactor, a cytokine, a chemokine, a hormone, an antibody, an antioxidant molecule, an engineered immunoglobulin-like molecule, a single-chain antibody, a fusion protein, an immune co-stimulatory molecule, an immunomodulatory molecule, a chimeric antigen receptor, a trans-domain negative mutant of a target protein, a toxin, a conditional toxin, an antigen, a transcription factor, a structural protein, a reporter protein, a subcellular localization signal, a tumor suppressor protein, a growth factor, a membrane protein, a receptor, a vasoactive protein or peptide, an antiviral protein or ribozyme, or a derivative thereof, or a microRNA.

24. The nucleotide of interest is: (i) Disorders responsive to: cytokine and cell proliferation / differentiation activity; immunosuppressant or immunostimulatory activity; modulation of hematopoiesis; promotion of growth of bone, cartilage, tendon, ligament and nerve tissue; inhibition or activation of follicle-stimulating hormone; chemotactic / chemokinetic activity; hemostatic and thrombolytic activity; anti-inflammatory activity; macrophage inhibitory and / or T-cell inhibitory activity and therefore anti-inflammatory activity; anti-immune activity; inhibition of the ability of macrophages and T-cells to adhere to extracellular matrix components and fibronectin, and upregulated fas receptor expression in T-cells; (ii) malignant disorders including cancer, leukemia, benign and malignant tumor growth, invasion and spread, angiogenesis, metastasis, ascites and malignant pleural effusion; (iii) autoimmune diseases, including arthritis, including rheumatoid arthritis, hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other diseases; (iv) vascular diseases, including arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome, cardiovascular effects, peripheral vascular disease, migraine and aspirin-dependent antithrombosis, stroke, cerebral ischemia, ischemic heart disease or other diseases; (v) diseases of the gastrointestinal tract, including peptic ulcers, ulcerative colitis, Crohn's disease and other diseases; (vi) liver disease, including liver fibrosis and cirrhosis; (vii) inherited metabolic disorders, including phenylketonuria (PKU), Wilson's disease, organic acidemias, urea cycle disorders, cholestasis, and other diseases; (viii) kidney and urinary tract diseases, including thyroiditis or other glandular diseases, glomerulonephritis or other diseases; (ix) ear, nose, and throat disorders, including otitis or other otorhinolaryngological diseases, dermatitis or other skin diseases; (x) dental and oral disorders, including periodontal disease, periodontitis, gingivitis or other dental / oral diseases; (xi) orchitis or epididymis - testicular disease, including orchitis, infertility, testicular trauma or other testicular disease; (xii) gynecological disorders, including placental insufficiency, placental failure, habitual miscarriage, eclampsia, preeclampsia, endometriosis, and other gynecological disorders; (xiii) Leber congenital amaurosis (LCA), including LCA10, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, glaucoma, including open-angle glaucoma and juvenile congenital glaucoma, intraocular inflammation, sympathetic ophthalmia, scleritis, retinitis pigmentosa, macular degeneration, including age-related macular degeneration (AMD) and juvenile macular degeneration, Best's disease, including Best's vitelliform macular degeneration, Stargardt's disease, Usher syndrome, Doyne honeycomb retinal dystrophy, Sorby macular dystrophy, juvenile glaucoma, and ocular malformations. Ophthalmologic disorders including age-related retinoschisis, cone-rod dystrophy, corneal dystrophies, Fuchs' dystrophy, Leber's congenital amaurosis, Leber's hereditary optic neuropathy (LHON), Adie's syndrome, Oguchi's disease, degenerative fundus diseases, ocular trauma, ocular inflammation caused by infection, proliferative vitreoretinopathy, acute ischemic optic neuropathy, excessive scarring, reactions to ocular implants, corneal graft rejection, and other ophthalmologic diseases including diabetic macular edema, retinal vein occlusion, RLBP1-related retinal dystrophies, choroideremia, and color vision disorders; (xiv) Parkinson's disease, complications and / or side effects of treatment for Parkinson's disease, AIDS-related dementia syndrome, HIV-associated encephalopathy, Devic's disease, Sydenham's chorea, Alzheimer's disease and other degenerative diseases, CNS conditions or disorders, stroke, post-polio syndrome, psychiatric disorders, myelitis, encephalitis, subacute sclerosing panencephalitis, encephalomyelitis, acute neurological disorders, subacute neurological disorders, chronic neurological disorders, Fabry's disease, Gaucher's disease, cystinosis, Pompe's disease, metachromatic leukodystrophy, Wiskott- Neurological and neurodegenerative disorders including Aldrich syndrome, adrenoleukodystrophy, beta thalassemia, sickle cell disease, Guillain-Barre syndrome, Sydenham chorea, myasthenia gravis, pseudotumor cerebri, Down's syndrome, Huntington's disease, CNS compression or CNS trauma or infection of the CNS, muscle atrophy and muscular dystrophies, diseases, conditions or disorders of the central and peripheral nervous system, amyotrophic lateral sclerosis, spinal muscular atrophy, and motor neuron diseases including spinal cord and avulsion injuries; and (xv) for suppressing or inhibiting humoral and / or cellular immune responses, or for the prevention and / or treatment of cystic fibrosis, mucopolysaccharidoses including Sanfilippo syndrome A, Sanfilippo syndrome B, Sanfilippo syndrome C, Sanfilippo syndrome D, Hunter syndrome, Hurler-Scheie syndrome, Morquio syndrome, ADA-SCID, X-linked SCID, X-linked chronic granulomatous disease, porphyria, hemophilia A, hemophilia B, post-traumatic inflammation, bleeding, coagulation and acute phase response, cachexia, anorexia, acute infections, septic shock, infections, diabetes, complications or side effects of surgery, complications and / or side effects of bone marrow transplantation or other transplants, complications and side effects of gene therapy, AIDS, or for the prevention and / or treatment of graft rejection in the case of transplantation of natural or artificial cells, tissues and organs, such as corneas, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue.

24. The cell of claim 23, wherein the cell encodes a molecule useful for treating a disorder selected from the group consisting of:

25. 25. The cell of claim 23 or 24, wherein the nucleotide of interest encodes a molecule useful for treating a disorder selected from immunodeficiency, human immunodeficiency virus infection, cancer, autoimmune disease, transplant rejection, myeloid disease, lymphoid disease, wounds, burns, ulcers, periodontal disease, neurodegeneration, infertility, hemophilia, stroke, septic shock and Crohn's disease.

26. A stable or transient production cell for producing a lentiviral vector comprising at least one nucleotide sequence encoding the modified U1 snRNA according to any one of claims 1 to 16.

27. The cell of any one of claims 19 to 26, wherein the lentiviral vector is derived from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or Visna lentivirus.

28. The cell of claim 27, wherein the lentiviral vector is derived from HIV-1, HIV-2, or EIAV.

29. 28. The cell of claim 27, wherein the lentiviral vector is derived from SIV.

30. a. Introducing into a cell a nucleotide sequence encoding vector components including gag, env, rev and the RNA genome of a lentiviral vector and at least one nucleotide sequence encoding the modified U1 snRNA of any one of claims 1 to 13; b. Optionally, selecting cells containing the vector components and the nucleotide sequence encoding at least one modified U1 snRNA; c. Culturing the cells under conditions in which the vector components are co-expressed with the modified U1 snRNA and a lentiviral vector is produced. A method for producing a lentiviral vector, comprising:

31. A lentiviral vector produced by the production method of claim 30.

32. 32. The lentiviral vector of claim 31, wherein the lentiviral vector is derived from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or visna lentivirus.

33. 33. The lentiviral vector of claim 32, wherein the lentiviral vector is derived from HIV-1, HIV-2, or EIAV.

34. 33. The lentiviral vector of claim 32, wherein the lentiviral vector is derived from SIV.

35. Use of a modified U1 snRNA according to any one of claims 1 to 16 or an expression cassette according to claim 17 for the production of a lentiviral vector.

36. 36. The use of claim 35, wherein the lentiviral vector comprises an inactivated major splice donor site in the RNA genome of the lentiviral vector.

37. 37. The use according to claim 35 or claim 36, wherein the lentiviral vector is derived from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or Visna lentivirus.

38. A cell according to any one of claims 19, 20 to 25 or 27 to 29, or a stable or transient production cell according to any one of claims 26 to 29, wherein the major splice donor site in the RNA genome of the lentiviral vector is inactivated.

39. The cell or stable or transient production cell of claim 38, wherein the major splice donor site and the cryptic splice donor site 3' to the major splice donor site in the RNA genome of the lentiviral vector are inactivated.

40. 39. The cell or stable or transient production cell of claim 38, wherein the lentiviral vector is a third generation lentiviral vector.

41. 39. The cell or stable or transient production cell of claim 38, wherein the major splice donor site in the RNA genome of the lentiviral vector is inactivated, and a cryptic splice donor site 3' to the major splice donor site is inactivated, and the nucleotide sequence is for use in a Tat-independent lentiviral vector.

42. The cell or stable or transient production cell of claim 38, wherein the major splice donor site in the RNA genome of the lentiviral vector is inactivated, a cryptic splice donor site 3' to the major splice donor site is inactivated, and the nucleotide sequence is produced in the absence of tat.

43. The cell or stable or transient production cell of claim 38, wherein the major splice donor site in the RNA genome of the lentiviral vector is inactivated, a cryptic splice donor site 3' to the major splice donor site is inactivated, and the nucleotide sequence is transcribed independently of tat.

44. The cell or stable or transient production cell of claim 38, wherein the major splice donor site in the RNA genome of the lentiviral vector is inactivated, and a cryptic splice donor site 3' to the major splice donor site is inactivated, and the nucleotide sequence is for use in a U3-independent lentiviral vector.

45. The cell or stable or transient production cell of claim 38, wherein the major splice donor site in the RNA genome of the lentiviral vector is inactivated, a cryptic splice donor site 3' to the major splice donor site is inactivated, and the nucleotide sequence is transcribed independently of the U3 promoter.

46. The cell or stable or transient production cell of claim 38, wherein the major splice donor site in the RNA genome of the lentiviral vector is inactivated, a cryptic splice donor site 3' to the major splice donor site is inactivated, and the nucleotide sequence is transcribed by a heterologous promoter.

47. 47. The cell or stable or transient production cell according to any one of claims 39 to 46, wherein the cryptic splice donor site is the first cryptic splice donor site 3' to the major splice donor site.

48. 48. The cell or stable or transient production cell of any one of claims 39, 41 to 47, wherein the cryptic splice donor site is located within 6 nucleotides of the major splice donor site.

49. 49. The cell or stable or transient production cell according to any one of claims 39, 41 to 48, wherein the major splice donor site and the cryptic splice donor site are mutated or deleted.

50. 50. The cell or stable or transient production cell of any one of claims 39, 41 to 49, wherein the nucleotide sequence encoding the RNA genome of the lentiviral vector before inactivation of the splice sites comprises a sequence set forth in any of SEQ ID NOs: 1, 3, 4, 9, 10 and / or 13.

51. 51. The cell or stable or transient production cell of any one of claims 39, 41 to 50, wherein the nucleotide sequence encoding the RNA genome of the lentiviral vector comprises a sequence containing a mutation or deletion compared to the sequence set forth in any of SEQ ID NOs: 1, 3, 4, 9, 10 and / or 13.

52. 52. The cell or stable or transient production cell of any one of claims 38 to 51, wherein the nucleotide sequence encoding the RNA genome of the lentiviral vector comprises an inactivated major splice donor site which, when not inactivated, has a cleavage site between the nucleotides corresponding to nucleotides 13 and 14 of SEQ ID NO:

1.

53. 53. The cell or stable or transient production cell according to any one of claims 38 to 52, wherein the nucleotide sequence of the major splice donor site before inactivation comprises the sequence set forth in SEQ ID NO:

4.

54. 54. The cell or stable or transient production cell according to any one of claims 39, 41 to 53, wherein the nucleotide sequence of the potential splice donor site before inactivation comprises the sequence set forth in SEQ ID NO:

10.

55. 55. The cell or stable or transient production cell of any one of claims 39 to 54, wherein the nucleotide sequence encoding the RNA genome of the lentiviral vector comprises an inactivated cryptic splice donor site which, when not inactivated, has a cleavage site between nucleotides corresponding to nucleotides 17 and 18 of SEQ ID NO:

1.

56. 56. The cell or stable or transient production cell of any one of claims 38 to 55, wherein the nucleotide sequence encoding the RNA genome of the lentiviral vector comprises a sequence set forth in any of SEQ ID NOs: 2, 5, 6, 7, 8, 11, 12 and / or 14.

57. The cell or stable or transient production cell according to any one of claims 38 to 56, wherein the nucleotide sequence encoding the RNA genome of the lentiviral vector does not include the sequence set forth in SEQ ID NO:

9.

58. A cell or stable or transient production cell according to any one of claims 38 to 57, in which splicing activity from the major splice donor site and cryptic splice donor sites of the RNA genome of the lentiviral vector is suppressed or eliminated.

59. A cell or stable or transient production cell according to any one of claims 38 to 58, wherein splicing activity from the major splice donor site and cryptic splice donor sites of the RNA genome of the lentiviral vector is suppressed or eliminated in the transfected or transduced cell.

60. 60. The cell of any one of claims 18, 20 to 25, 27 to 29 or 38 to 59, or the stable or transient production cell of any one of claims 26 or 38 to 59, wherein a nucleotide sequence encoding the RNA genome of the lentiviral vector is operably linked to a nucleotide sequence encoding a modified U1 snRNA.

61. a. the major splice donor site in the RNA genome of the lentiviral vector is inactivated, and / or b) The method of claim 30, wherein the major splice donor site and the cryptic splice donor site 3' to the major splice donor site in the RNA genome of the lentiviral vector are inactivated.

62. a. The major splice donor site and the cryptic splice donor site are mutated or deleted; b. The nucleotide sequence encoding the RNA genome of the lentiviral vector comprises the sequence set forth in any of SEQ ID NOs: 2, 5, 6, 7, 8, 11, 12, and / or 14; c. The nucleotide sequence encoding the RNA genome of the lentiviral vector does not include the sequence set forth in SEQ ID NO:9; d. splicing activity from the major splice donor site and cryptic splice donor sites of the RNA genome of the lentiviral vector is suppressed or eliminated; and / or e. Splicing activity from the major splice donor site and cryptic splice donor site of the RNA genome of the lentiviral vector is suppressed or eliminated in transfected or transduced cells; The method of claim 61.

63. 31. The method of claim 30, wherein the nucleotide sequence encoding the RNA genome of the lentiviral vector is operably linked to a nucleotide sequence encoding the modified U1 snRNA.

64. The use according to claim 36 or 37, wherein the major splice donor site and the cryptic splice donor site 3' to the major splice donor site in the RNA genome of the lentiviral vector are inactivated.

65. a. The major splice donor site and the cryptic splice donor site are mutated or deleted; b. The nucleotide sequence encoding the RNA genome of the lentiviral vector comprises the sequence set forth in any of SEQ ID NOs: 2, 5, 6, 7, 8, 11, 12, and / or 14; c. The nucleotide sequence encoding the RNA genome of the lentiviral vector does not include the sequence set forth in SEQ ID NO:9; d. splicing activity from the major splice donor site and cryptic splice donor sites of the RNA genome of the lentiviral vector is suppressed or eliminated; and / or e. Splicing activity from the major splice donor site and cryptic splice donor site of the RNA genome of the lentiviral vector is suppressed or eliminated in transfected or transduced cells; 38. The use according to claim 37.

66. 38. The use according to claim 37, wherein the nucleotide sequence encoding the RNA genome of the lentiviral vector is operably linked to a nucleotide sequence encoding the modified U1 snRNA.

67. 17. A lentiviral vector produced in the presence of the modified U1 snRNA of any one of claims 1 to 16, wherein the lentiviral vector comprises an inactivated major splice donor site in the RNA genome of the lentiviral vector.

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