Production System

Abstract

The present invention relates to a nucleic acid sequence comprising a nucleotide of interest and a tryptophan RNA-binding attenuation protein (TRAP) binding site, and optionally a Kozak sequence, wherein the TRAP binding site overlaps the Kozak sequence and / or the ATG start codon of the nucleotide of interest. The present invention further relates to a nucleic acid sequence comprising a nucleotide of interest and a Kozak sequence, wherein the Kozak sequence comprises a portion of the tryptophan RNA-binding attenuation protein (TRAP) binding site. The present invention further relates to a nucleic acid sequence comprising a nucleotide of interest and a TRAP binding site, wherein the TRAP binding site comprises a portion of the ATG start codon of the nucleotide of interest, or wherein the ATG start codon comprises a portion of the TRAP binding site. The present invention further relates to a nucleic acid sequence comprising a nucleotide of interest, a binding site for tryptophan RNA-binding attenuation protein (TRAP), a multiple cloning site, and a Kozak sequence, wherein the multiple cloning site overlaps the 3'KAGN2-3 repeat of the TRAP binding site or is located downstream of the 3'KAGN2-3 repeat of the TRAP binding site and upstream of the Kozak sequence.

Description

[Technical Field]

[0001] The present invention relates to the production of viral vectors. More specifically, the present invention relates to the modification of the translation of a nucleotide of interest encoded by a viral vector in a viral vector producing cell. [Background technology]

[0002] Gene therapy broadly involves the use of genetic material to treat disease. Gene therapy includes replacing a functional copy of a defective gene (e.g., a gene with a mutation) in cells that have the defective gene, inactivating an improperly functioning gene, and introducing a new therapeutic gene.

[0003] Therapeutic genetic material can be incorporated into target cells of the host using vectors to allow for the transfer of nucleic acids. Such vectors can generally be divided into viral and non-viral categories.

[0004] Viruses naturally introduce their genetic material into host target cells as part of their replication cycle. Engineered viral vectors exploit this ability to deliver a nucleotide of interest (NOI) or transgene to target cells. To date, many viruses have been engineered as vectors for gene therapy. These include retroviruses, adenoviruses (AdV), adeno-associated viruses (AAV), herpes simplex viruses (HSV), and vaccinia viruses.

[0005] In addition to being modified to carry NOI, viral vectors are typically further engineered to be replication-defective.Therefore, recombinant vectors can directly infect target cells, but cannot produce further generations of infectious virions.Other types of viral vectors can be conditionally replicative only in cancer cells, and can further encode toxic transgenes or proenzymes.

[0006] Retroviral vectors are being developed as treatments for a variety of genetic disorders and are currently showing increasing promise in clinical trials (e.g., Galy, A. and A. J. Thrasher (2010) Curr Opin Allergy Clin Immunol 11(6):545-550; Porter, DL, BL Levine, M. Kalos, A. Bagg and C. H. June (2011) N Engl J Med 365(8):725-733; Campochiaro, PA (2012) Gene Ther 19(2):121-126; Cartier, N., S. Hacein-Bey-Abina, C. C. Bartholomae, P. Bougneres, M. Schmidt, C. V. Kalle, A. Fischer, M. Cavazzana-Calvo and P. Aubourg (2012) Methods Enzymol 507:187-198;Sadelain, M., I. Riviere, X. Wang, F. Boulad, S. Prockop, P. Giardina, A. Maggio, R. Galanello, F. Locatelli and E. Yannaki (2010) Ann NY Acad Sci. 1202:52-58;DiGiusto,DL,A.Krishnan,L.Li,H.Li,S.Li,A.Rao,S.Mi,P.Yam,S.Stinson,M.Kalos,J.Alvarnas,SFLacey,JKYee,M.Li,L.Couture,D.Hsu,SJForman,JJRossi and JAZaia(2010)Sci Transl Med 2(36):36ra43 and Segura MM, MM, Gaillet B, Garnier A. (2013)Expert opinion in biological therapy).

[0007] Important examples of such vectors include gamma-retroviral vector systems (based on MMLV), primate lentiviral vector systems (based on HIV-1) and non-primate lentiviral vector systems (based on EIAV).

[0008] Reverse genetics has made it possible to significantly engineer these virus-based vectors so that vectors encoding large heterologous sequences (approximately 10 kb) can be produced by transfecting mammalian cells with the appropriate DNA sequence (reviewed in Bannert, K. (2010) Caister Academic Press: 347-370).

[0009] The manipulation and use of retroviral vectors in research typically involves the production of reporter gene vectors encoding, for example, GFP or lacZ. The titers of these clinically irrelevant vectors are typically around 1 x 10 per mL of crude harvest material. 6 ~1×10 7 Further concentration and purification of this material is in the region of 1 x 10 transducing units (TU / mL). 10 Working stocks of greater than TU / mL can be achieved, however production of vectors encoding therapeutically relevant NOIs often results in substantially reduced titres compared to these reporter vectors.

[0010] There are several factors that may be contributing to this effect.

[0011] 1. Size of the therapeutic genome. Although very large genomes can be packaged by retroviruses, the reverse transcription and / or integration steps are thought to become less efficient as size increases.

[0012] 2. Stability of the vector genomic RNA, which can be reduced by the presence of unexpected destabilizing elements in the NOI.

[0013] 3. Suboptimal nucleotide usage within the vector genome RNA. Wild-type viral genomes often have certain nucleotide biases (e.g., HIV-1 is AT-rich). Vector genomes tend to be less AT-rich, which may affect packaging and / or post-maturation processes.

[0014] 4. Expression of the NOI in viral vector producer cells. The (over)expressed protein may have an indirect or direct effect on the assembly and / or infectivity of the vector virion.

[0015] Experience has shown that expression of proteins encoded by NOIs in viral vector producing cells can adversely affect the titer of therapeutic vectors (as described in WO 2015 / 092440).

[0016] Incorporation of the protein encoded by the NOI (protein of interest, POI) into the vector virion can also affect downstream processing of the vector particle; for example, an NOI encoding a transmembrane POI can result in high surface expression of the transmembrane protein in the viral vector virion, potentially altering the physical properties of the virion. Furthermore, this incorporation can present the POI to the patient's immune system at the delivery site, which can adversely affect transduction and / or long-term expression of the therapeutic gene in vivo. NOIs can also induce the production of undesirable secondary proteins or metabolites that can affect production, purification, recovery, and immunogenicity, and it is desirable to minimize this.

[0017] The ability to suppress NOI expression in viral vector-producing cells while maintaining effective expression of the NOI in target cells is also desirable. Whatever mechanism is used, the "natural" pathway of viral vector particle assembly and resulting functionality must not be disrupted. This is not straightforward, since the viral vector genome molecule packaged into virions must necessarily encode an NOI expression cassette. In other words, because the vector genome molecule and the NOI expression cassette are operably linked, modifications to the NOI expression cassette may adversely affect the ability to produce the vector genome molecule within the cell. For example, using a physical transcription blocker (e.g., the TetR repressor system) to suppress the NOI expression cassette is likely to also inhibit the production of the vector genome molecule due to steric hindrance. Furthermore, modifications to the regulatory mechanisms must also not adversely affect the functionality of the vector genome molecule after virion maturation and release (i.e., with respect to inducing transduction of target cells). For example, retroviral vector genome RNA molecules must be capable of the processes of reverse transcription and integration, and any modifications to the NOI expression cassette must not interfere with these steps in the transduction process.

[0018] Suppression of NOI expression in viral vector-producing cells may offer additional advantages. If NOI expression results in reduced viability of vector-producing cells, its suppression may benefit large-scale production, which requires large cell numbers. Reducing cell debris due to cell death also reduces impurities in the crude vector harvest material. It may be possible to standardize the processing, purification, and concentration of vector platforms (i.e., different therapeutic genes encoded within the same vector system); if the only heterologous genes expressed in viral vector-producing cells were those required for vector production, downstream processing could be more easily optimized for the entire therapeutic vector platform, resulting in highly similar physical specifications of the vector preparations. Variability in the immune response to the resulting vector in vivo and toxicity of the resulting vector may be minimized, which may result in more sustained therapeutic NOI expression in target cells.

[0019] Tissue-specific promoters that limit expression of the NOI in producer cells are a possible solution to this problem, but the leakiness of these promoters can result in deleterious levels of transgene protein. However, constitutive promoters can be used to achieve greater and more robust expression of the NOI in target cells. Indeed, such robust expression may be required for in vivo efficacy. Furthermore, tissue-specific promoters may be less predictable when observing therapeutic vector products through animal models and into humans during preclinical and clinical development.

[0020] WO 2015 / 092440 (hereby incorporated by reference) discloses the use of a heterologous translational control system in eukaryotic cell culture to repress translation of an NOI (repress transgene expression) during viral vector production, thereby repressing or preventing expression of the protein encoded by the NOI. This system allows for transgene repression ( T ransgene R EpressionI n vector P This system is called the transgene induction cell system (TRIP system). In one form, the TRIP system utilizes the bacterial trp operon regulatory proteins, tryptophan RNA-binding attenuation protein (TRAP) and the TRAP binding site / sequence (tbs) to mediate transgene silencing. Surprisingly, use of this system does not interfere with the production of packageable vector genome molecules or the activity of vector virions, and does not interfere with long-term expression of the NOI in target cells. [Prior art documents] [Patent documents]

[0021] [Patent Document 1] International Publication No. 2015 / 092440 [Non-patent literature]

[0022] [Non-Patent Document 1] Galy, A. and AJ Thrasher (2010) Curr Opin Allergy Clin Immunol 11(6):545-550 [Non-patent document 2] Porter, DL, BLLevine, M. Kalos, A. Bagg and CH June (2011) N Engl J Med 365(8):725-733 [Non-patent document 3] Campochiaro, PA (2012) Gene Ther 19(2):121-126 [Non-patent document 4] Cartier, N., S. Hacein-Bey-Abina, CC Bartholomae, P. Bougheres, M. Schmidt, CV Kalle, A. Fischer, M. Cavazzana-Calvo and P. Aubourg (2012) Methods Enzymol 507:187-198 [Non-Patent Document 5] Sadelain, M., I. Riviere, X. Wang, F. Boulad, S. Prockop, P. Giardina, A. Maggio, R. Galanello, F. Locatelli and E. Yannaki (2010) Ann NY Acad Sci 1202:52-58 [Non-patent document 6] DiGiusto,DL,A.Krishnan,L.Li,H.Li,S.Li,A.Rao,S.Mi,P.Yam,S.Stinson,M.Kalos,J.Alvarnas,SFLacey,JKYee,M.Li,L.Couture,D.Hsu,SJForman,JJRossi and JAZaia(2010)Sci Transl Med 2(36):36ra43 [Non-Patent Document 7] Segura MM, MM, Gaillet B, Garnier A. (2013) Expert opinion in biological therapy [Non-patent document 8] Bannert, K. (2010) Caister Academic Press: 347-370 Summary of the Invention [Means for solving the problem]

[0023] The present invention relates to modifications made to transgene mRNA that can be used to improve the TRAP system, allowing for improved levels of translational repression by TRAP. The improved nucleic acid sequences of the present invention can have, for example, the following characteristics:

[0024] 1. We show that an improved 5'UTR leader sequence (upstream of tbs) composed of nucleotides derived from the first (non-coding) exon of the EF1α gene can surprisingly enable consistently lower "repressed" levels of transgene expression mediated by the TRAP-tbs complex compared to 5'UTR leader sequences from various constitutive promoters.

[0025] 2. An improved UTR or "spacer" sequence inserted between the internal ribosome entry site (IRES) and the tbs is surprisingly shown to improve both fold repression and non-repression levels (i.e., no TRAP).

[0026] 3. A mutant Kozak sequence overlapping the 3' end of tbs is surprisingly shown to result in improved occlusion of the transgene start codon by the TRAP-tbs complex.

[0027] 4. We show that sequences containing compressed, overlapping multiple cloning sites between tbs and the transgene Kozak sequence (transgene start codon (ATG)) can surprisingly facilitate cloning while retaining low levels of transgene expression when suppressed by TRAP.

[0028] 5. It is shown that overlap of the 3' end of tbs with the transgene start codon ATG surprisingly results in improved occlusion of the transgene start codon by the TRAP-tbs complex.

[0029] In one aspect, the present invention provides a nucleic acid sequence comprising a nucleotide of interest, a tryptophan RNA-binding attenuation protein (TRAP) binding site, and a Kozak sequence, wherein the TRAP binding site overlaps with the Kozak sequence.

[0030] In another aspect, the present invention provides a nucleic acid sequence comprising a nucleotide of interest and a Kozak sequence, wherein the Kozak sequence comprises a portion of a tryptophan RNA-binding attenuation protein (TRAP) binding site.

[0031] In one aspect, the present invention provides a nucleic acid sequence comprising a nucleotide of interest (transgene) and a TRAP binding site, wherein the TRAP binding site comprises a portion of the transgene start codon ATG, or vice versa.

[0032] In some embodiments, the nucleotide of interest is operably linked to a TRAP binding site or portion thereof.

[0033] In some embodiments, the TRAP binding site or a portion thereof can interact with a tryptophan RNA-binding attenuating protein such that translation of the nucleotide of interest is repressed in the viral vector-producing cell.

[0034] In some embodiments, the nucleotide of interest is translated in a target cell that lacks a tryptophan RNA-binding attenuating protein.

[0035] In some embodiments, the TRAP binding site or portion thereof comprises multiple repeats of the sequence KAGN2-3.

[0036] In some embodiments, the TRAP binding site or portion thereof comprises multiple repeats of the sequence KAGN2.

[0037] In some embodiments, the TRAP binding site or portion thereof comprises at least six repeats of the sequence KAGN2.

[0038] In some embodiments, the TRAP binding site or portion thereof comprises at least eight repeats of the sequence KAGN2-3. The number of KAGNNN repeats may be one or less.

[0039] In some embodiments, the TRAP binding site or portion thereof comprises at least 8-11 repeats of the sequence KAGN2.

[0040] In some embodiments, the TRAP binding site or portion thereof comprises 11 repeats of the sequence KAGN2-3, and the number of KAGNNN repeats is 3 or less.

[0041] In some embodiments, the Kozak sequence overlaps the 3' end of or a portion of the TRAP binding site. The Kozak sequence may overlap the 3' terminal KAGNN repeat of or a portion of the TRAP binding site.

[0042] In some embodiments, the Kozak sequence comprises the sequence RNNATG.

[0043] In some embodiments, the Kozak sequence comprises the sequence RVVATG.

[0044] In some embodiments, the overlapping Kozak sequence and TRAP binding site or portion thereof has the following sequence: (a) GAGATG; (b) KAGVATG; (c)KAGVVATG; (d) KAGRVVATG; or (e)KAGNRVVATG Includes one of the following.

[0045] In some embodiments, the nucleic acid sequence is the following sequence: (a) KAGCCGAGATG; (b) KAGGCGAGCATG; (c)KAGNGGAGCCATG; or (d)KAGNNGAGACCATG Includes one of the following.

[0046] In some embodiments, the nucleic acid sequence is the following sequence: (a) KAGCCGAGATG; or (b)KAGNGGAGCCATG Includes one of the following.

[0047] In some embodiments, the nucleic acid sequence comprises the sequence set forth in SEQ ID NOs: 69-92 or 108-112.

[0048] In some embodiments, the distance from the end of the transcription start site / promoter to the start of the TRAP binding site or portion thereof is less than 34 nucleotides.

[0049] In some embodiments, the distance from the end of the transcription start site / promoter to the start of the TRAP binding site or portion thereof is less than 13 nucleotides.

[0050] In some embodiments, the TRAP binding site or portion thereof lacks a type II restriction enzyme site, preferably a SapI restriction enzyme site.

[0051] In some embodiments, the nucleic acid sequence comprises a 5' leader sequence upstream of the TRAP binding site or a portion thereof. The leader sequence may comprise a sequence derived from the non-coding EF1α exon 1 region. The leader sequence may comprise the sequence defined in SEQ ID NO:25 or SEQ ID NO:26.

[0052] In some embodiments, the nucleic acid sequence comprises an internal ribosome entry site (IRES). The nucleic acid sequence may comprise a spacer sequence between the internal ribosome entry site (IRES) and the TRAP binding site or a portion thereof. The spacer may be 0 to 30 nucleotides in length. The spacer may be 15 nucleotides in length.

[0053] In some embodiments, the spacer is 3 or 9 nucleotides from the 3' end of the TRAP binding site or portion thereof and downstream of the start codon of the nucleotide of interest.

[0054] In some embodiments, the spacer comprises the sequence defined in any one of SEQ ID NOs: 38-44, preferably the spacer comprises the sequence defined in SEQ ID NO: 38.

[0055] In some embodiments, the nucleotide of interest produces a therapeutic effect.

[0056] In some embodiments, the nucleic acid sequence further comprises an RRE sequence or a functional substitute thereof.

[0057] In some embodiments, the nucleic acid sequence is a vector transgene expression cassette.

[0058] In some embodiments, the 3' terminal KAGNN repeat of or portion of the TRAP binding site overlaps with at least the first nucleotide of the ATG start codon.

[0059] In some embodiments, the 3' terminal KAGNN repeat of or a portion of the TRAP binding site overlaps the first two nucleotides of the ATG start codon.

[0060] In some embodiments, the 3' terminal KAGNN repeat of or a portion of the TRAP binding site overlaps the first nucleotide of the ATG start codon within the core Kozak sequence as defined herein.

[0061] In some embodiments, the nucleic acid sequence comprises the sequence defined in SEQ ID NO:114 or SEQ ID NO:116.

[0062] In a further aspect, the present invention provides a viral vector comprising a nucleic acid sequence of the present invention.

[0063] In some embodiments, the viral vector comprises more than one nucleotide of interest, and at least one nucleotide of interest is operably linked to a TRAP binding site or portion thereof as defined herein.

[0064] In some embodiments, the viral vector is derived from retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus or baculovirus.The viral vector can be derived from lentivirus.The viral vector can be derived from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or visna lentivirus.

[0065] In a further aspect, the present invention provides a viral vector production system comprising a set of nucleic acid sequences encoding components required for the production of a viral vector, wherein the vector genome comprises the nucleic acid sequences of the present invention. The viral vector may be derived from a retrovirus, an adenovirus, or an adeno-associated virus.

[0066] In some embodiments, the viral vector is a retroviral vector and the viral vector production system further comprises nucleic acid sequences encoding Gag and Pol proteins, a tryptophan RNA-binding attenuator protein, and an Env protein, or functional substitutes thereof.

[0067] In some embodiments, the viral vector production system further comprises a nucleic acid sequence encoding rev or a functional substitute thereof.

[0068] In some embodiments, the viral vector is derived from a lentivirus. The viral vector can be derived from an HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or visna lentivirus.

[0069] In a further aspect, the present invention provides a DNA construct for use in the viral vector production system of the present invention, comprising a nucleic acid sequence of the present invention.

[0070] In a further aspect, the present invention provides a DNA construct for use in the viral vector production system of the present invention, comprising a nucleic acid sequence encoding a tryptophan-RNA binding attenuating protein.

[0071] In a further aspect, the present invention provides a set of DNA constructs for use in the viral vector production system of the present invention, comprising the DNA construct of the present invention, DNA constructs encoding Gag and Pol proteins, and a DNA construct encoding an Env protein or a functional substitute thereof.

[0072] In some embodiments, the set of DNA constructs further comprises a DNA construct encoding the rev sequence or a functional substitute thereof.

[0073] In a further aspect, the present invention provides a viral vector-producing cell comprising a nucleic acid sequence of the present invention and a viral vector-producing system of the present invention or a DNA construct of the present invention.

[0074] In some embodiments, the cells are transiently transfected with a vector encoding a tryptophan-RNA-binding attenuating protein. The cells may stably express the tryptophan-RNA-binding attenuating protein.

[0075] In a further aspect, the present invention provides a method for producing a viral vector, comprising introducing the nucleic acid sequence of the present invention, the viral vector production system of the present invention, or the DNA construct of the present invention into a viral vector producing cell, and culturing the producer cell under conditions suitable for producing the viral vector.

[0076] In a further aspect, the present invention provides a viral vector produced by the viral vector production system of the present invention, using the viral vector producing cell of the present invention or by the method of the present invention.

[0077] In some embodiments, the viral vector comprises a nucleic acid sequence of the invention.

[0078] In some embodiments, the viral vector is derived from a retrovirus, adenovirus, or adeno-associated virus. The viral vector can be derived from a lentivirus. The viral vector can be derived from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or visna lentivirus.

[0079] In a further aspect, the present invention provides a cell transduced with a viral vector of the present invention.

[0080] In a further aspect, the present invention provides a viral vector of the invention or a cell of the invention for use in medicine.

[0081] In a further aspect, the present invention provides use of the viral vector of the present invention or the cell of the present invention for the preparation of a medicament for delivering a nucleotide of interest to a target site requiring the nucleotide of interest.

[0082] In a further aspect, the present invention provides a method of treatment comprising administering the viral vector of the present invention or the cell of the present invention to a subject in need thereof.

[0083] In a further aspect, the present invention provides a pharmaceutical composition comprising a viral vector of the present invention or a cell of the present invention in combination with a pharmaceutically acceptable carrier, diluent or excipient.

[0084] In a further aspect, the present invention provides a method for identifying a nucleic acid binding site and / or a nucleic acid binding protein that can interact such that when operably linked to the nucleic acid binding site, translation of a nucleotide of interest is suppressed in a viral vector producing cell, the method comprising analyzing expression of a reporter gene in a cell containing both the nucleic acid binding site and the nucleic acid binding protein operably linked to the reporter gene.

[0085] In some embodiments, the reporter gene encodes a fluorescent protein.

[0086] In a further aspect, the present invention provides a method for suppressing translation of a nucleotide of interest (NOI) in a viral vector producing cell, the method comprising introducing into the viral vector producing cell a nucleic acid sequence of the present invention and a nucleic acid sequence encoding a tryptophan-RNA binding attenuating protein (TRAP), wherein the TRAP binds to the TRAP binding site or a portion thereof, thereby suppressing translation of the NOI.

[0087] In a further aspect, the present invention provides a method for increasing viral vector titer in a eukaryotic vector-producing cell, the method comprising introducing into the eukaryotic vector-producing cell a viral vector production system of the present invention and a nucleic acid sequence encoding a tryptophan-RNA-binding attenuation protein (TRAP), wherein the TRAP binds to the TRAP binding site or a portion thereof and inhibits translation of the NOI, thereby increasing the viral vector titer compared to a viral vector that does not have a TRAP binding site.

[0088] In a further aspect, the present invention provides a nucleic acid sequence comprising a nucleotide of interest, a binding site for tryptophan RNA-binding attenuation protein (TRAP), a multiple cloning site, and a Kozak sequence, wherein the multiple cloning site is located downstream of the TRAP binding site and upstream of the Kozak sequence.

[0089] In some embodiments, the nucleic acid sequence of the present invention further comprises a promoter. The TRAP binding site or a portion thereof and a Kozak sequence, or the TRAP binding site, multiple cloning site, and a Kozak sequence, may be located within the 5'UTR of the promoter.

[0090] In some embodiments, the promoter further comprises an intron, preferably the intron is upstream of the TRAP binding site or a portion thereof. The promoter may be an engineered promoter containing a heterologous intron within the 5'UTR.

[0091] In some embodiments, the nucleic acid sequence of the present invention comprises the sequence set forth in any of SEQ ID NOs: 117, 118, or 120-124.

[0092] In a further aspect, the present invention provides a nucleic acid sequence encoding the RNA genome of a viral vector, wherein the RNA genome of the viral vector comprises a nucleic acid sequence described herein.

[0093] In some embodiments, the nucleic acid sequences of the invention described herein are contained within the RNA genome of a viral vector.

[0094] In some embodiments, the nucleic acid sequences of the invention described herein are operably linked to a nucleotide sequence encoding the RNA genome of a viral vector.

[0095] In some embodiments of the nucleic acid sequences of the invention described herein or the viral vector production systems of the invention described herein, the major splice donor site in the RNA genome of the viral vector is inactivated.

[0096] In some embodiments, the major splice donor site and a cryptic splice donor site 3' to the major splice donor site in the RNA genome of the viral vector are inactivated.

[0097] In some embodiments, the cryptic splice donor site is the first cryptic splice donor site 3' to the major splice donor site.

[0098] In some embodiments, the cryptic splice donor site or sequence is within 6 nucleotides of the major splice donor site or sequence.

[0099] In some embodiments, the major splice donor site and the cryptic splice donor sites are mutated or deleted.

[0100] In some embodiments, the nucleotide sequence encoding the RNA genome of the viral vector prior to inactivation of splice sites comprises the sequence set forth in any of SEQ ID NOs: 94, 96, 97, 102, 103 and / or 106.

[0101] In some embodiments, the nucleotide sequence encoding the RNA genome of the viral vector comprises a sequence having a mutation or deletion relative to the sequence set forth in any of SEQ ID NOs: 94, 96, 97, 102, 103 and / or 106.

[0102] In some embodiments, the nucleotide sequence encoding the RNA genome of the viral vector comprises an inactivated major splice donor site that has a cleavage site between nucleotides that would otherwise correspond to nucleotides 13 and 14 of SEQ ID NO:94.

[0103] In some embodiments, the nucleotide sequence of the major splice donor site before inactivation comprises the sequence set forth in SEQ ID NO:97.

[0104] In some embodiments, the nucleotide sequence of the cryptic splice donor site before inactivation comprises the sequence set forth in SEQ ID NO:103.

[0105] In some embodiments, the nucleotide sequence encoding the RNA genome of the viral vector contains an inactivated cryptic splice donor site that would otherwise have a cleavage site between nucleotides corresponding to nucleotides 17 and 18 of SEQ ID NO:94.

[0106] In some embodiments, the nucleotide sequence encoding the RNA genome of the viral vector comprises the sequence set forth in any of SEQ ID NOs: 95, 98, 99, 100, 101, 104, 105 and / or 107.

[0107] In some embodiments, the nucleotide sequence encoding the RNA genome of the viral vector does not include the sequence set forth in SEQ ID NO:102.

[0108] In some embodiments, splicing activity from major and cryptic splice donor sites in the RNA genome of the viral vector is inhibited or eliminated.

[0109] In some embodiments, splicing activity from major and cryptic splice donor sites in the RNA genome of the viral vector is suppressed or eliminated in transfected or transduced cells.

[0110] In some embodiments, the viral vector is derived from a lentivirus. [Brief explanation of the drawings]

[0111] [Figure 1] Overview of improvements to the 5' UTR sequence upstream of tbs. Schematic diagram showing the location of the 5' leader sequence from exon 1 of the EF1α gene, placed upstream of the tbs sequence ([KAGNN]x11) within the transgene 5' UTR. Surprisingly, when compared with leader sequences from other promoters, the use of such a leader sequence is found to result in improved levels of transgene repression by TRAP-tbs (TRAP represented by a doughnut shape). Without wishing to be bound by theory, it is presumed that such a leader sequence allows for a more stable TRAP-tbs complex, such that inhibition of ribosome scanning is maximized. [Figure 2]Summary of improvements to the 5' UTR sequence downstream of tbs. A. Schematic diagram showing the DNA expression cassette of the 5' UTR coding region of a TRAP-tbs suppressive transgene cassette in which a multiple cloning site (MCS) is inserted between tbs and the start codon of the transgene (TRAP is represented by a donut shape). The present invention describes preferred overlapping restriction enzyme sites that begin at / on the terminal KAGNN repeat of tbs and contain up to five cloning sites upstream of the transgene start codon. B. Schematic diagram showing how the Kozak sequence of the transgene can be positioned to largely or partially overlap the 3' KAGNN repeat of tbs; doing so effectively "hides" the major start codon in the TRAP-tbs complex, making it less accessible to the translational machinery and resulting in a lower, "suppressed" level of transgene expression. C. Table summarizing preferred overlapping tbs and Kozak consensus sequences. The 3′ KAGNN repeat of tbs is shown in a box, and the core Kozak sequence is shown in bold. [Figure 3]Enhanced repression by TRAP-tbs using the L33-improved leader sequence compared to various constitutive promoters containing the native UTR sequence. A. Schematic diagram showing the construction of the GFP test reporter plasmid. The 5'UTR contained the same tbs sequence and other elements, except for the different promoters utilized, as well as different leader sequences upstream of the tbs (these are shown in panel I of Table I). Note that, due to the absence of intron sequences in the mRNAs, the intron-containing EF1α (EF1a) and UBC promoters are considered directly comparable to their "short" intronless counterparts, EFS and UBC, respectively (as per Table I). A 34-nt leader was present in the CMV promoter-containing reporter as a control (previously shown to enable over 100-fold repression by TRAP-tbs). Other constitutive promoters were engineered in this study to contain leaders containing several synthetic sequences in addition to the native leader sequence, or to have the L33-improved leader sequence derived from exon 1 of the EF1α promoter. B. The reporter was tested for unrepressed or repressed levels of GFP expression by cotransfection of the reporter plasmid with either pBlueScript (no TRAP) or pEF1α-TRAP (TRAP), respectively. Transfected HEK293T cells (suspension, serum-free) were analyzed by flow cytometry 2 days after transfection to obtain a GFP expression score (% GFP × median fluorescence intensity), which was log-10 transformed. Data in the chart are displayed by general promoter strength (no TRAP levels) from left to right, compared to mock-transfected cells (pBlueScript only) or untransfected cells (UNT). In each case where the native leader was compared to the L33-improved leader, the L33-improved leader allowed for substantially lower levels of GFP expression in the presence of TRAP, allowing for a more than 10-fold improvement over repression by TRAP-tbs. In many cases, the unrepressed levels of expressed GFP were also slightly improved when the L33-improved leader was used. (standard deviation bars, n=3). [Figure 4]Design and evaluation of multiple cloning sites (MCSs) inserted between the tbs and the Kozak sequence of transgene cassettes within the AAV vector genome, and their effect on transgene silencing by TRAP-tbs. A. Schematic showing the MCS variants tested along with the 5'UTR-tbs sequence; seven variants contain two to four cloning sites without an NcoI site, depending on the presence of a particular Kozak sequence and the first nucleotide of the second codon of the transgene (and therefore may not be present in all transgene cassettes). The MCS variant reporter constructs were driven by the EFS promoter and contained the L33-improved leader, whereas the "no MCS" control reporter construct was driven by the CMV promoter and had the original 34-nt leader (shown in Figure 4 to function similarly to the L33-improved leader). The transgene cassettes were cloned into scAAV2 vector genome plasmids (ITRs not shown). B. Reporter AAV genome plasmids were tested for unrepressed or repressed levels of GFP expression by cotransfection of the reporter plasmid with either pBlueScript (no TRAP) or pEF1a-TRAP (TRAP), respectively. Transfected HEK293T cells (suspension, serum-free) were analyzed by flow cytometry 2 days after transfection, and GFP expression scores (% GFP × median fluorescence intensity) were obtained and log-10 transformed. All MCS mutant reporters were repressed approximately 1000-fold or more by TRAP-tbs, and six of seven mutants were at least 10-fold better at reducing transgene levels. Furthermore, mutants MCS2.1, MCS4.1, and MCS4.4 enabled TRAP-tbs to repress GFP levels (compared to untransfected [UNT]) to the limit of detection (standard deviation bars, n=3). [Figure 5]We demonstrated consistent and robust transgene suppression by TRAP-tbs using transgene cassettes driven by different constitutive promoters with an L33-improved leader, tbs, and an optimized multiple cloning site (MCS4.1-GFP) sequence. Various constitutive promoters were cloned into the MCS2.1-GFP and MCS4.1-GFP scAAV2 reporter genome plasmids containing the L33-tbs sequence. The reporters were tested for unrepressed or repressed levels of GFP expression by cotransfecting the reporter plasmids with either pBlueScript (no TRAP) or pEF1α-TRAP (TRAP), respectively. Transfected HEK293T cells (suspension, serum-free) were analyzed by flow cytometry 2 days after transfection, and GFP expression scores (% GFP × median fluorescence intensity) were obtained and log-10 transformed. The data demonstrate that the L33-improved leader and optimized MCS sites can be incorporated into the same cassette, enabling high transgene suppression by TRAP-tbs for a wide range of constitutive promoters. The average level of inhibition across the entire experiment was approximately 5000-fold (standard deviation bars, n=3). [Figure 6]Identification of an optimal Kozak sequence overlapping the 3' end of tbs within the transgene 5' UTR to position tbs closer to the ATG start codon. A. Schematic showing the location and sequence of Kozak sequences in engineered variants conforming to the core consensus "RVVATG" and the broader consensus "GNNRVVATG," in which Kozak sequences were positioned to overlap the 3' end of tbs so that the KAGNN repeat(s) were maintained. This allowed for the placement of tbs closer to the ATG start codon to identify tbs-Kozak junction variants that would allow improved transgene suppression levels (+TRAP) by "hiding" the ATG start codon within the TRAP-tbs complex. Maintenance of the consensus Kozak sequence allowed for the retention of high levels of unsuppressed transgenes (without TRAP) (i.e., modeling expression in cells transduced with the vector). B. The reporters were tested for unrepressed or repressed levels of GFP expression by cotransfection of the reporter plasmid with either pBlueScript (no TRAP) or pEF1α-TRAP (TRAP) into HEK293T cells, respectively. Transfected cells were analyzed by flow cytometry 2 days after transfection, and a GFP expression score (% GFP × median fluorescence intensity) was obtained and log-10 transformed. All tbs-Kozak junction mutant reporters maintained identical, unrepressed GFP levels compared to the original construct. Mutants "0," "2," and "3" showed improved repression levels compared to the original construct (standard deviation bars, n = 3). [Figure 7-1]Identification of an improved spacer sequence between the IRES and tbs sequences to confer better repression of IRES-dependent transgenes by TRAP-tbs. A. Schematic showing the configuration of the transgene cassette when testing spacer sequences. The pCMV-luciferase-IRES-(spacer)-tbs-GFP reporter construct was designed (see Table III) and tested. B. Reporters containing the original [26 nt] spacer or mutant [26 nt] or two spacer truncations were tested. The reporters were tested for unrepressed or repressed levels of GFP expression by cotransfection of the reporter plasmid with either pBlueScript (no TRAP) or pEF1α-TRAP (TRAP) into HEK293T cells, respectively. Transfected cells were analyzed by flow cytometry 2 days after transfection, and a GFP expression score (% GFP × median fluorescence intensity) was obtained and log-10 transformed. This study revealed the "original [trunc-15nt]" spacer as a variant with improved "ON" and reduced "OFF" levels compared to the original spacer. The "original [trunc-15nt]" spacer was then incorporated into reporters with either an 11xKAGNN repeat tbs or an 8xKAGNN repeat tbs, at the 3' end of the tbs and either 9 nt or 3 nt distance from the downstream transgene ATG start codon. GFP expression was measured as previously described. The data demonstrate that the improved "original [trunc-15nt]" spacer can be used with different tbs configurations and proximity to the primary transgene ATG start codon (standard deviation bars, n = 3). [Figure 7-2]Identification of an improved spacer sequence between the IRES and tbs sequences to confer better repression of IRES-dependent transgenes by TRAP-tbs. A. Schematic showing the configuration of the transgene cassette when testing spacer sequences. The pCMV-luciferase-IRES-(spacer)-tbs-GFP reporter construct was designed (see Table III) and tested. B. Reporters containing the original [26 nt] spacer or mutant [26 nt] or two spacer truncations were tested. The reporters were tested for unrepressed or repressed levels of GFP expression by cotransfection of the reporter plasmid with either pBlueScript (no TRAP) or pEF1α-TRAP (TRAP) into HEK293T cells, respectively. Transfected cells were analyzed by flow cytometry 2 days after transfection, and a GFP expression score (% GFP × median fluorescence intensity) was obtained and log-10 transformed. This study revealed the "original [trunc-15nt]" spacer as a variant with improved "ON" and reduced "OFF" levels compared to the original spacer. The "original [trunc-15nt]" spacer was then incorporated into reporters with either an 11xKAGNN repeat tbs or an 8xKAGNN repeat tbs, at the 3' end of the tbs and either 9 nt or 3 nt distance from the downstream transgene ATG start codon. GFP expression was measured as previously described. The data demonstrate that the improved "original [trunc-15nt]" spacer can be used with different tbs configurations and proximity to the primary transgene ATG start codon (standard deviation bars, n = 3). [Figure 8]Comparison of two improved leaders derived from the EF1α exon 1 sequence. The truncated leader "L12" was derived from the L33 improved leader sequence, which includes exon 1 from the human EF1α gene (see Table I). The L12 improved leader was cloned into six constitutive promoter-containing GFP reporter cassettes within scAAV2 vector genome plasmids, carrying either the MCS2.1 or MCS4.1 sequence between the tbs and Kozak sequences. The reporters were tested for unrepressed or repressed levels of GFP expression by cotransfection of the reporter plasmid with either pBlueScript (no TRAP) or pEF1α-TRAP (TRAP), respectively. Transfected HEK293T cells (suspension, serum-free) were analyzed by flow cytometry 2 days after transfection, and a GFP expression score (% GFP × median fluorescence intensity) was obtained and log-10 transformed. The data demonstrate that the L12 and L33-improved leaders allow complete repression by TRAP-tbs, suppressing GFP levels to background levels. Interestingly, GFP "ON" (unrepressed) levels are slightly higher for L12 or L33 under different promoters, allowing flexibility in that either L12 or L33 can be selected when considering the use of the TRAP system with different promoters, thereby maximizing gene expression levels in the absence of TRAP (i.e., in cells transduced with the vector) without losing the substantial level of repression achieved by TRAP-tbs during vector production. (Standard deviation bars, n=3). [Figure 9]Improved transgene silencing in AAV vector genome plasmids using overlapping tbs-Kozak variants. Two "tbs-Kozak" variants (0 and 3) were cloned into either the EFS or huPGK promoter-GFP reporter cassettes, which additionally contained either the L33 or L12-improved leader sequence. Non-overlapping tbs / Kozak variants were also cloned into the EFS / huPGK-L33 cassette; these differed only in the tbs-Kozak region (original = [tbs]-ACAGCCACCATG; HpaI variant = [tbs-GAGTT]AACGCCACCATG). The reporters were tested for unrepressed or repressed levels of GFP expression by cotransfection of the reporter plasmid with either pBlueScript (without TRAP) or pEF1α-TRAP (TRAP), respectively. Transfected HEK293T cells (suspension, serum-free) were analyzed by flow cytometry 2 days after transfection to obtain a GFP expression score (% GFP × median fluorescence intensity) that was log-10 transformed. The data demonstrate that overlapping the tbs with the Kozak sequence allows for improved suppression of transgene expression by TRAP compared to the non-overlapping tbs / Kozak variant. (Standard deviation bars, n=3). [Figure 10-1]Improved TRAP-mediated transgene silencing in the context of the full-length EF1α promoter. A. Three "tbs-Kozak" variants (0, 2, and 3) were cloned into the EF1α promoter-GFP reporter cassette. After splicing, the leader sequence contains the L33 sequence (exon 1) and a short 12-nt sequence from exon 2 immediately upstream of the tbs. B. The reporter was tested for unrepressed or repressed levels of GFP expression by cotransfection of the reporter plasmid with either pBlueScript (no TRAP) or pEF1α-TRAP (TRAP), respectively. Transfected HEK293T cells (suspension, serum-free) were analyzed by flow cytometry 2 days after transfection, and a GFP expression score (% GFP × median fluorescence intensity) was obtained and log-10 transformed. C. The GFP transgene cassette was cloned into an HIV-1 lentiviral vector genome and tested for unrepressed or repressed levels of GFP expression as described in B. The data demonstrate that overlapping the tbs with the Kozak sequence allows for improved repression of transgene expression by TRAP (standard deviation bars, n=3). [Figure 10-2]Improved TRAP-mediated transgene silencing in the context of the full-length EF1α promoter. A. Three "tbs-Kozak" variants (0, 2, and 3) were cloned into the EF1α promoter-GFP reporter cassette. After splicing, the leader sequence contains the L33 sequence (exon 1) and a short 12-nt sequence from exon 2 immediately upstream of the tbs. B. The reporter was tested for unrepressed or repressed levels of GFP expression by cotransfection of the reporter plasmid with either pBlueScript (no TRAP) or pEF1α-TRAP (TRAP), respectively. Transfected HEK293T cells (suspension, serum-free) were analyzed by flow cytometry 2 days after transfection, and a GFP expression score (% GFP × median fluorescence intensity) was obtained and log-10 transformed. C. The GFP transgene cassette was cloned into an HIV-1 lentiviral vector genome and tested for unrepressed or repressed levels of GFP expression as described in B. The data demonstrate that overlapping the tbs with the Kozak sequence allows for improved repression of transgene expression by TRAP (standard deviation bars, n=3). [Figure 11]During lentiviral vector production, aberrantly spliced ​​mRNAs expressing transgenes are eliminated in MSD-2KO lentiviral vectors, reducing the amount of transgene mRNA required 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, with a TRAP binding site (tbs) located within the cassette's 5'UTR (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 predominant forms of cytoplasmic 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 transgenes (ii), which can occur independently of the internal transgene promoter, i.e., tissue-specific promoter. (Key: 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 {f} and reverse {r} primers 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. 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 generated (%GFP x MFI). Compared to the total amount of GFP produced in culture during standard lentiviral vector production, the MSD-2KO modification had a substantial effect (approximately 5-fold) 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, resulting in much lower levels of GFP in the cultures. C The sequence of the stem-loop 2 (SL2) region of "wild-type" HIV-1 (NL4-3; the "standard" sequence in the current lentiviral vector genome) is shown above. The sequence includes a major splice donor site (MSD: consensus = CTGGT) and a cryptic splice donor site (used when the MSD site itself is mutated (crSD: consensus = TGAGT). Nucleotides at the splicing positions when the splice donor site is used are identified by bold letters and arrows. Four functional MSD mutations that abolish both MSD and crSD site splicing activity are described: 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 a mutation that abolishes both the MSD and crSD sites; MSD-2KOm5, which introduces an entirely new stem-loop structure lacking any 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 shown in lowercase italics. [Figure 12]The impact of aberrant splicing from the major splice donor site (MSD) within HIV-1-based lentiviral vectors. Standard third-generation lentiviral vectors were produced in HEK293T cells with + / - rev, and total RNA was extracted from the cells after production. Total RNA was subjected to qPCR (SYBR Green) using two primer sets: f+rT, which amplifies total transcripts generated from the lentiviral vector expression cassette, and f+rUS, which amplifies 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 standard third-generation lentiviral vector production is low and varies according to the internal transgene cassette (in this case, containing a different promoter and GFP gene); furthermore, this ratio is only minimally increased by the effect of rev. [Figure 13-1]TRAP-mediated transgene suppression of overlapping tbs-Kozak mutants was tested in suspension (serum-free) HEK293T cells. The overlapping tbs-Kozak mutants in Table IV were cloned into the pEF1a-GFP reporter plasmid and transfected into HEK293T cells + / -pTRAP. Flow cytometry was performed 2 days posttransfection. A. GFP expression scores (% GFP positivity × MFI) and fold suppression values ​​were determined and plotted in + / -TRAP. Mutants are shown along the x-axis and grouped according to the relative overlap of the 3' tbs KAGNN repeat and core Kozak sequence ("overlap group" - KAGNN, KAGNN, KAGNN); KAGNN is represented by a black bracket, and the core Kozak nucleotide is represented by a gray line. Statistical analyses were performed comparing the following overlap groups (equal variances within overlap groups were confirmed by F-test): using a two-tailed T-test, fold suppression was statistically greater for KAGatg relative to KAGNatg (*p=0.0293), for KAGNNatg relative to KAGNatg (**p=0.00000482), and for KAGNNatg relative to non-overlapping tbs (***p=0.000259). B. Unsuppressed GFP expression scores are plotted from highest to lowest (left to right), highlighting the two KAGatg overlap group mutants tbskzkV0.G and tbskzkV0.T (showing the greatest suppression among all mutants in A) to demonstrate that the "G" mutant is preferred over the "T" mutant because it has superior "ON" (unsuppressed) levels. [Figure 13-2]TRAP-mediated transgene suppression of overlapping tbs-Kozak mutants was tested in suspension (serum-free) HEK293T cells. The overlapping tbs-Kozak mutants in Table IV were cloned into the pEF1a-GFP reporter plasmid and transfected into HEK293T cells + / -pTRAP. Flow cytometry was performed 2 days posttransfection. A. GFP expression scores (% GFP positivity × MFI) and fold suppression values ​​were determined and plotted in + / -TRAP. Mutants are shown along the x-axis and grouped according to the relative overlap of the 3' tbs KAGNN repeat and core Kozak sequence ("overlap group" - KAGNN, KAGNN, KAGNN); KAGNN is represented by a black bracket, and the core Kozak nucleotide is represented by a gray line. Statistical analyses were performed comparing the following overlap groups (equal variances within overlap groups were confirmed by F-test): using a two-tailed T-test, fold suppression was statistically greater for KAGatg relative to KAGNatg (*p=0.0293), for KAGNNatg relative to KAGNatg (**p=0.00000482), and for KAGNNatg relative to non-overlapping tbs (***p=0.000259). B. Unsuppressed GFP expression scores are plotted from highest to lowest (left to right), highlighting the two KAGatg overlap group mutants tbskzkV0.G and tbskzkV0.T (showing the greatest suppression among all mutants in A) to demonstrate that the "G" mutant is preferred over the "T" mutant because it has superior "ON" (unsuppressed) levels. [Figure 14-1]Improved suppression of intron-containing promoters using optimal overlapping tbs-Kozak variants. A. Schematic diagram of the expression cassette used to illustrate the use of overlapping tbs-Kozak variants compared to non-overlapping tbs-Kozak variants. The widely used EF1a promoter sequence, like the widely used CAG promoter, contains its own intron (see Figure 10 and Example 5). The CAG promoter is an extremely powerful artificial promoter containing a CMV enhancer, a core promoter, and exon 1 / intron sequences from the chicken β-actin gene and a splice acceptor / exon sequence from the rabbit β-globin gene. In this study, the "EF1a-INT" sequence from the EF1a promoter (containing exon 1 [L33]), all of the EF1a intron and splice acceptor, and 12 nucleotides from EF1a exon 2 were cloned into the CAG promoter, replacing the CAG exon / intron sequence. The "EF1a-INT" sequence was also cloned into the CMV promoter construct. B. To model transgene expression during viral vector production, constructs were evaluated for GFP expression and repression by TRAP in suspension (serum-free) HEK293T cells. GFP expression scores (% GFP x MFI) were calculated and plotted, along with fold repression scores in the presence of TRAP. [Figure 14-2]Improved suppression of intron-containing promoters using optimal overlapping tbs-Kozak variants. A. Schematic diagram of the expression cassette used to illustrate the use of overlapping tbs-Kozak variants compared to non-overlapping tbs-Kozak variants. The widely used EF1a promoter sequence, like the widely used CAG promoter, contains its own intron (see Figure 10 and Example 5). The CAG promoter is an extremely powerful artificial promoter containing a CMV enhancer, a core promoter, and exon 1 / intron sequences from the chicken β-actin gene and a splice acceptor / exon sequence from the rabbit β-globin gene. In this study, the "EF1a-INT" sequence from the EF1a promoter (containing exon 1 [L33]), all of the EF1a intron and splice acceptor, and 12 nucleotides from EF1a exon 2 were cloned into the CAG promoter, replacing the CAG exon / intron sequence. The "EF1a-INT" sequence was also cloned into the CMV promoter construct. B. To model transgene expression during viral vector production, constructs were evaluated for GFP expression and repression by TRAP in suspension (serum-free) HEK293T cells. GFP expression scores (% GFP x MFI) were calculated and plotted, along with fold repression scores in the presence of TRAP. DETAILED DESCRIPTION OF THE INVENTION

[0112] Various preferred features and aspects of the present invention will now be described by way of non-limiting example.

[0113] 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. For example, J. Sambrook, EFFritsch, and T. Maniatis (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, FMet 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;JMPolak and James O'D.McGee(1990)In Situ Hybridization:Principles and Practice;Oxford University Press;MJGait(ed.)(1984)Oligonucleotide Synthesis:A Practical Approach,IRL Press;and,DMJLilley and JEDahlberg(1992)Methods See "Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology," Academic Press, 1999. Each of these general texts is incorporated herein by reference.

[0114] The present disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of aspects of the present disclosure. Numerical ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequence is written left to right in the 5' to 3' direction, and amino acid sequences are written left to right in the amino to carboxy direction, respectively.

[0115] Where a range of values ​​is given, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range is also specifically disclosed, unless the context clearly dictates otherwise. 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 encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded, and each range in which either, neither, or both limits are included in the smaller range is also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. When a stated range includes one or both of the limits, ranges excluding either or both of the included limits are also included in the disclosure.

[0116] Please note 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.

[0117] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," or "containing," "contains," and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps. The terms "comprising," "comprises," and "comprised of" also include the term "consisting of."

[0118] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0119] Nucleic acid sequence In one aspect, the present invention provides a nucleic acid sequence comprising a nucleotide of interest, a tryptophan RNA-binding attenuation protein (TRAP) binding site, and a Kozak sequence, wherein the TRAP binding site (tbs) overlaps with the Kozak sequence.

[0120] In one aspect, the present invention provides a nucleic acid sequence comprising a nucleotide of interest (transgene) and a tryptophan RNA-binding attenuation protein (TRAP) binding site (tbs), wherein the TRAP binding site overlaps with the transgene start codon ATG.

[0121] In another aspect, the present invention provides a nucleic acid sequence comprising a nucleotide of interest and a Kozak sequence, wherein the Kozak sequence comprises a portion of a tryptophan RNA-binding attenuation protein (TRAP) binding site (tbs).

[0122] In one aspect, the present invention provides a nucleic acid sequence comprising a nucleotide of interest (transgene) and a TRAP binding site, wherein the TRAP binding site (tbs) comprises a portion of the transgene start codon ATG, or vice versa.

[0123] In some embodiments of the present invention, the nucleotide of interest is operably linked to the tbs or a portion thereof. In some embodiments, the nucleotide of interest is translated in a target cell that lacks TRAP.

[0124] The tbs or a portion thereof may be capable of interacting with TRAP such that translation of the nucleotide of interest is suppressed or prevented in viral vector producing cells.

[0125] Therefore, in another aspect, the present invention provides a method for suppressing translation of NOI in a viral vector producing cell, comprising introducing a nucleic acid sequence of the present invention and a nucleic acid sequence encoding TRAP into a viral vector producing cell, wherein TRAP binds to the TRAP binding site or a portion thereof, thereby suppressing translation of NOI.

[0126] Tryptophan RNA-binding decay protein (TRAP) Tryptophan RNA-binding decay protein (TRAP) is a bacterial protein that has been extensively characterized in Bacillus subtilis. TRAP regulates tryptophan biosynthesis directed by the trpEDCFBA operon by participating in either transcriptional attenuation or translational control mechanisms (reviewed in Gollnick, B., Antson, and Yanofsky (2005) Annual Review of Genetics 39:47-68).

[0127] In its natural context, TRAP regulates tryptophan biosynthesis and transport by three different mechanisms.

[0128] 1. Attenuation of transcription of the trpEDCFBA operon (Shimotsu H, KM, Yanofsky C, Henner DJ. (1986) Journal of Bacteriology 166:461-471).

[0129] 2. Promotion of the formation of the trpE and trpD Shine-Dalgarno-blocking hairpins (Yakhnin H, BJ, Yakhnin AV, Babitzke P. (2001) Journal of Bacteriology 183(20):5918-5926).

[0130] 3. Blocking ribosome access to the trpG and yhaG ribosome binding sites (Yang M, dSA, van Loon APGM, Gollnick P. (1995) Journal of Bacteriology 177:4272-4278).

[0131] In Bacillus subtilis, TRAP is encoded by a single gene (mtrB), and the functional protein consists of 11 identical subunits arranged as a toroidal ring (Antson AA, DE, Dodson G, Greaves RB, Chen X, Gollnick P. (1999) Nature 401(6750):235-242). TRAP is activated and interacts with RNA by binding up to 11 tryptophan molecules in a pocket between adjacent subunits. The target RNA is wrapped around the outside of this quaternary ring structure (Babitzke P, SJ, Shire SJ, Yanofsky C. (1994) Journal of Biological Chemistry 269:16597-16604).

[0132] Without wishing to be bound by theory, it is believed that in the natural mechanism for sensing and controlling tryptophan synthesis, TRAP acts at the level of transcription termination by binding to a binding site in the newly synthesized RNA leader. This destabilizes the overlapping antiterminator sequence, resulting in the production of only short RNAs, so that the downstream rho-independent terminator is active. When tryptophan is limiting in bacteria, the TRAP loop can no longer bind to its RNA binding site. Therefore, the antiterminator is activated, and transcription continues into the tryptophan synthesis gene operon. TRAP can also act at the translational level: tryptophan-dependent binding of TRAP to its binding site in the 5'-UTR of the RNA transcript releases the anti-Shine-Dalgarno sequence, which forms a stable stem with the Shine-Dalgarno sequence, thereby inhibiting ribosome initiation of translation. Finally, in other situations where TRAP is bound to its tbs, it can inhibit translation initiation by physically sequestering the 40S scanning ribosomal complex before it can reach the start codon and allow the otherwise more stable, higher affinity translation machinery to immediately form.

[0133] Because bacterial gene sequences are likely not optimal for expression in mammalian cells, the TRAP open reading frame can be codon-optimized for expression in mammalian (e.g., Homo sapiens) cells. The sequence can also be optimized by removing potential unstable sequences and splice sites. The use of a C-terminal HIS tag on the TRAP protein appears to provide benefits for translational repression and may be used. This C-terminal HIS tag may improve TRAP solubility or stability in eukaryotic cells, but the improved functional benefits cannot be eliminated. Nevertheless, both HIS-tagged and untagged TRAP enabled robust repression of transgene expression. Certain cis-acting sequences within the TRAP transcription unit can also be optimized; for example, constructs driven by the EF1α promoter allow better repression at lower inputs of the TRAP plasmid compared to constructs driven by the CMV promoter in transient transfection situations.

[0134] In one embodiment, the TRAP is derived from bacteria.

[0135] In one embodiment of the present invention, the TRAP is derived from a Bacillus species, such as Bacillus subtilis. For example, the TRAP may comprise the following sequence:

[0136] MNQKHSSDFVVIKAVEDGVNVIGLTRGTDTKFHHSEKLDKGEVIIAQFTEHTSAIKVRGEALIQTAYGEMKSEKK (SEQ ID NO: 1) In a preferred embodiment of the invention, SEQ ID NO: 1 is tagged at the C-terminus with six histidine amino acids (HISx6 tag).

[0137] In an alternative embodiment, the TRAP is derived from Aminomonas paucivorans. For example, the TRAP may comprise the following sequence:

[0138] MKEGEEAKTSVLSDYVVVKALENGVTVIGLTRGQETKFAHTEKLDDGEVWIAQFTEHTSAIKVRGASEIHTKHGMLFSGRGRNEKG (SEQ ID NO: 2) In an alternative embodiment, the TRAP is derived from Desulfotomaculum hydrothermal. For example, the TRAP may comprise the following sequence:

[0139] MNPMTDRSDITGDYVVVKALENGVTIIGLTRGGVTKFHHTEKLDKGEIMIAQFTEHTSAIKIRGRAELLTKHGKIRTEVDS (SEQ ID NO: 3) In an alternative embodiment, the TRAP is derived from B. stearothermophilus. For example, the TRAP may comprise the following sequence:

[0140] MYTNSDFVVIKALEDGVNVIGLTRGADTRFHHSEKLDKGEVLIAQFTEHTSAIKVRGKAYIQTRHGVIESEGKK (SEQ ID NO: 4) In an alternative embodiment, the TRAP is derived from B. stearothermophilus S72N. For example, the TRAP may comprise the following sequence:

[0141] MYTNSDFVVIKALEDGVNVIGLTRGADTRFHHSEKLDKGEVLIAQFTEHTSAIKVRGKAYIQTRHGVIENEGKK (SEQ ID NO: 5) In an alternative embodiment, the TRAP is derived from B. halodurans. For example, the TRAP may comprise the following sequence:

[0142] MNVGDNSNFFVIKAKENGVNVFGMTRGTDTRFHHSEKLDKGEVMIAQFTEHTSAVKIRGKAIIQTSYGTLDTEKDE (SEQ ID NO: 6) In an alternative embodiment, the TRAP is derived from Carboxydothermus hydrogenoformans. For example, the TRAP may comprise the following sequence:

[0143] MVCDNFAFSSAINAEYIVVKALENGVTIMGLTRGKDTKFHHTEKLDKGEVMVAQFTEHTSAIKIRGKAEIYTKHGVIKNE (SEQ ID NO: 7) In one embodiment, TRAP is encoded by the tryptophan RNA-binding decay protein gene family mtrB (eg, TrpBP superfamily with domain database #cl03437 deposited in NCBI).

[0144] In a preferred embodiment, the TRAP is tagged at the C-terminus with six histidine amino acids (HISx6 tag).

[0145] In a preferred embodiment, the TRAP has 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% identity to any of SEQ ID NOs: 1 to 7 and comprises an amino acid sequence capable of interacting with an RNA binding site such that expression of the functionally linked NOI is modified, e.g., suppressed or prevented, in a viral vector producing cell.

[0146] In a preferred embodiment, the TRAP has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% identity to any of SEQ ID NOs: 1 to 7 and comprises an amino acid sequence capable of interacting with an RNA binding site such that expression of the functionally linked NOI is modified, e.g., suppressed or prevented, in a viral vector producing cell.

[0147] In another embodiment, a TRAP may be encoded by a polynucleotide comprising a nucleotide sequence encoding a protein capable of interacting with an RNA-binding site such that expression of the operably linked NOI is modified, e.g., suppressed or prevented, in a viral vector-producing cell. For example, a TRAP may be encoded by a polynucleotide comprising a nucleotide sequence encoding a protein of SEQ ID NO: 1 to 7.

[0148] All variants, fragments or homologues of TRAP for use in the present invention retain the ability to bind to the TRAP binding site described herein, such that translation of the NOI (which may be a marker gene) is suppressed or prevented in viral vector producing cells.

[0149] TRAP binding site The term "binding site" should be understood as a nucleic acid sequence that is able to interact with a particular protein.

[0150] The consensus TRAP binding site sequence capable of binding TRAP is repeated multiple times (e.g., 6, 7, 8, 9, 10, 11, 12 or more times) [KAGN], and such sequences are found in the natural trp operon. In the natural context, AAGNN is sometimes tolerated, and sometimes additional "spacing" N nucleotides result in a functional sequence. In vitro experiments have demonstrated that at least six or more consensus repeats are required for TRAP-RNA binding (Babitzke P, YJ, Campanelli D. (1996) Journal of Bacteriology 178(17):5159-5163). Therefore, preferably, in one embodiment, six or more consecutive [KAGN] repeats are present within the trp operon. ≧2 ] sequence is present, where K can be T or G in DNA and U or G in RNA.

[0151] In the case of TRAP as the RNA-binding protein, the TRAP system preferably functions maximally with a tbs sequence containing at least eight KAGNN repeats, although seven repeats can still be used to obtain robust transgene suppression, and six repeats can be used to sufficiently suppress the transgene to a level that can rescue vector titer. The KAGNN consensus sequence can be altered to maintain TRAP-mediated suppression, but the exact sequence selected can preferably be optimized to ensure high levels of translation in the unsuppressed state. For example, the tbs sequence can be optimized by removing splice sites, unstable sequences, or stem-loops that may interfere with mRNA translation efficiency in the absence of TRAP (i.e., in target cells). For a given tbs KAGNN repeat configuration, the number of N "spacing" nucleotides between KAG repeats is preferably two. However, tbs containing more than two N spacers between at least two KAG repeats can be tolerated (as judged by in vitro binding studies, as many as 50% of three N-containing repeats can result in functional tbs; Babitzke P, YJ, Campanelli D. (1996) Journal of Bacteriology 178(17):5159-5163). Indeed, it has been shown that the 11×KAGNN tbs sequence can tolerate up to three replacements with KAGNNN repeats and still retain some potentially useful translation-blocking activity in conjunction with TRAP binding.

[0152] In one embodiment of the invention, the TRAP binding site or a portion thereof has the sequence KAGN ≧2 (e.g., KAGN 2~3 ) for the avoidance of doubt, this tbs or part thereof includes, for example, any of the following repeat sequences: UAGNN, GAGNN, TAGNN, UAGNNN, GAGNNN or TAGNNN.

[0153] "N" should be understood to designate any nucleotide at that position in the sequence. For example, it can be G, A, T, C, or U. The number of such nucleotides is preferably 2, but can be up to 3, e.g., 1, 2, or 3, and the KAG repeats of the 11x repeat tbs or a portion thereof can be separated by 3 spacing nucleotides and still retain some TRAP binding activity that results in translational repression. Preferably, no more than one N3 spacer will be used in the 11x repeat tbs or a portion thereof to retain maximum TRAP binding activity that results in translational repression.

[0154] In another embodiment, the tbs or a portion thereof is ≧2 Multiple iterations of (e.g., KAGN 2~3 (including multiple repetitions of

[0155] In another embodiment, the tbs or portion thereof comprises multiple repeats of the sequence KAGN2.

[0156] In another embodiment, the tbs or a portion thereof is ≧2 At least six iterations of (e.g., KAGN 2~3 at least six repeats of

[0157] In another embodiment, the tbs or a portion thereof comprises at least six repeats of KAGN2. For example, the tbs or a portion thereof can comprise 6, 7, 8, 9, 10, 11, 12 or more repeats of KAGN2. For example, the tbs or a portion thereof can comprise any one of SEQ ID NOs: 8-19 or 22.

[0158] In another embodiment, the tbs or a portion thereof is ≧2 At least 8 iterations of (e.g., KAGN 2~3 For example, the tbs or a portion thereof may include any one of SEQ ID NOs: 8, 9, 14-17, and 20-24.

[0159] Preferably, the number of KAGNNN repeats present in the tbs or portion thereof is 1 or less. For example, the tbs or portion thereof may comprise any one of SEQ ID NOs: 8, 9, 14-17, 19-24.

[0160] In another embodiment, the tbs or a portion thereof is ≧2 11 iterations of (e.g., KAGN 2~3 Preferably, the number of KAGNNN repeats present in this tbs or portion thereof is 3 or fewer. For example, the tbs or portion thereof may comprise any one of SEQ ID NOs: 8, 9, 14, 20-22.

[0161] In another embodiment, the tbs or a portion thereof is ≧2 12 iterations of (e.g., KAGN 2~3 (12 repetitions of the

[0162] In a preferred embodiment, the tbs or a portion thereof comprises 8 to 11 repeats of KAGN2 (e.g., 8, 9, 10, or 11 repeats of KAGN2). For example, the tbs or a portion thereof can comprise any one of SEQ ID NOs: 8, 9, 14-17, 20-24.

[0163] In one embodiment, the TRAP binding site or a portion thereof may comprise any of SEQ ID NOs: 8-24.

[0164] For example, the TRAP binding site or portion thereof may comprise any of the following sequences:

[0165] GAGUUUAGCGGAGUGGAGAAGAGCGGAGCCGAGCCUAGCAGAGACGAGUGGAGCU (SEQ ID NO: 8); or GAGUUUAGCGGAGUGGAGAAGAGCGGAGCCGAGCCUAGCAGAGACGAGAAGAGCU (SEQ ID NO: 9) "KAGN ≧2 "Iteration" is a general KAGN ≧2 (e.g., KAGN 2~3It should be understood that the ) motif is repeated. Different KAGNs that meet the criteria of this motif ≧2 Sequences can be linked to form a tbs or a portion thereof. It is not intended that the resulting tbs or portion thereof be limited to a single repeat of a sequence that meets the requirements of this motif, but this possibility is included in the definition. For example, "KAGN ≧2 "Six repeats of" include, but are not limited to, the following sequences: UAGUU-UAGUU-UAGUU-UAGUU-UAGUU-UAGUU (SEQ ID NO: 10); UAGUU-UAGUU-GAGUU-UAGUU-GAGUU-UAGUU (SEQ ID NO: 11); GAGUUU-GAGUU-GAGUU-GAGUUU-GAGUU-GAGUU (SEQ ID NO: 12) and UAGUUU-GAGUU-UAGUU-GAGUUU-UAGUU-GAGUU (SEQ ID NO: 13) (The dashes are included here between the repeats for clarity only).

[0166] An 8-repeat tbs sequence or a portion thereof containing one KAGNNN repeat and seven KAGNNN repeats retains TRAP-mediated repression activity. A tbs sequence or a portion thereof containing less than 8 repeats (e.g., a 7- or 6-repeat tbs sequence or a portion thereof) containing one or more KAGNNN repeats may have lower TRAP-mediated repression activity. Therefore, when there are fewer than 8 repeats, it is preferred that the tbs sequence or a portion thereof contain only KAGNNN repeats.

[0167] Preferred nucleotides for use in the KAGNN repeat consensus are as follows:

[0168] a pyrimidine in at least one of the N-N spacer positions; NN pyrimidine at the first spacer position; · Pyrimidines in both N-N spacer positions; ·G in K position.

[0169] It is also preferred that when the NN spacer position is AA, G is used at the K position (ie, TAGAA is preferably not used as a repeat in the consensus sequence).

[0170] By "capable of interacting" it should be understood that the nucleic acid binding site (e.g., tbs or a portion thereof) is capable of binding to a protein, such as TRAP, under conditions encountered in a cell, e.g., a eukaryotic viral vector producing cell. Such interaction with an RNA binding protein, such as TRAP, results in the repression or prevention of translation of the NOI to which the nucleic acid binding site (e.g., tbs or a portion thereof) is operably linked.

[0171] "Operably linked" should be understood as meaning that the described components are in a relationship permitting the described components to function in their intended manner. Thus, a tbs or portion thereof for use in the present invention operably linked to an NOI is positioned such that when a TRAP binds to the tbs or portion thereof, translation of the NOI is modified.

[0172] Placing a tbs or portion thereof capable of interacting with TRAP upstream of the NOI translation start codon of a given open reading frame (ORF) enables specific translational repression of mRNA derived from that ORF. The number of nucleotides separating the tbs or portion thereof from the translation start codon can vary, for example, from 0 to 34 nucleotides, without affecting the degree of repression. As a further example, 0 to 13 nucleotides can be used to separate the TRAP binding site or portion thereof from the translation start codon.

[0173] The tbs or a portion thereof can be placed downstream of an internal ribosome entry site (IRES) to suppress the translation of NOIs in multicistronic mRNAs. Indeed, this provides further evidence that TRAP bound to the tbs can block the passage of 40S ribosomes; the IRES element functions to corral the 40S ribosomal subunit to the mRNA in a CAP-independent manner before the complete translation complex is formed (for a review of IRES translation initiation, see Thompson, S. (2012) Trends in Microbiology 20(11):558-566). Therefore, the TRIP system can suppress multiple open reading frames from a single mRNA expressed from a viral vector genome. This is a useful feature of the TRIP system when creating vectors encoding multiple therapeutic genes, especially when all introduced gene products can adversely affect the vector titer to some extent.

[0174] In one embodiment, the nucleic acid sequence comprises a spacer sequence between the IRES and the tbs or a portion thereof. The IRES may be an IRES described herein under the subheading "Internal Ribosome Entry Site." The spacer sequence may be 0 to 30 nucleotides in length, preferably 15 nucleotides in length. The spacer may comprise a sequence defined in any one of SEQ ID NOs: 38 to 44, and preferably, the spacer comprises a sequence defined in SEQ ID NO: 38.

[0175] In one embodiment the spacer sequence between the IRES and the tbs or portion thereof is 3 or 9 nucleotides from the 3' end of the tbs or portion thereof and downstream start codon of the NOI.

[0176] In one embodiment, the tbs or portion thereof lacks a type II restriction enzyme site. In a preferred embodiment, the tbs or portion thereof lacks a SapI restriction enzyme site.

[0177] In some embodiments, the nucleic acid sequence further comprises an RRE sequence or a functional substitute thereof.

[0178] In some embodiments, the nucleic acid sequence is a vector transgene expression cassette.

[0179] Overlapping Kozak sequences and TRAP binding sites The present inventors surprisingly found that improved levels of suppression could be achieved by "hiding" the Kozak sequence within the 3' end of the tbs or a portion thereof (using overlapping tbs and Kozak sequences; see Figures 2B and 2C) compared with the use of non-overlapping tbs and Kozak sequences. Furthermore, unexpectedly, all of the overlapping Kozak and tbs sequences tested induced efficient levels of translation initiation, i.e., the overlapping sequences tested provided similar levels of transgene expression to non-overlapping Kozak and tbs sequences in the absence of TRAP. Without wishing to be bound by theory, the improved level of suppression may be due to improved occlusion of the transgene start codon by the TRAP-tbs complex when the tbs or a portion thereof overlaps with the Kozak sequence.

[0180] The term "Kozak sequence" should be understood as a consensus sequence in eukaryotic mRNA that is recognized by ribosomes as a translation initiation site. The Kozak sequence contains the ATG initiation (start) codon in DNA (AUG in mRNA). The exact Kozak sequence present in eukaryotic mRNA determines the efficiency of translation initiation; some Kozak sequences do not result in efficient translation initiation.

[0181] A complete Kozak sequence is typically understood to have the consensus sequence (gcc)gccRccATGG for DNA and (gcc)gccRccAUGG for RNA, where: lowercase letters represent the most common base at this position where the base can vary; uppercase letters represent highly conserved bases at this position; "R" typically indicates that a purine (i.e., A or G) is optimal at this position; and the sequence in parentheses (gcc) is of uncertain significance. T / U is generally the least preferred nucleotide at all positions in the Kozak sequence consensus upstream of the start codon.

[0182] Because the importance of the first three bases of a complete Kozak sequence is uncertain, the Kozak sequence can also be understood to have a consensus sequence referred to herein as the "extended Kozak sequence," i.e., GNNRVVATGG (SEQ ID NO: 27) for DNA and GNNRVVAUGG for RNA, where "R" should be understood to designate a purine (i.e., A or G) at that position in the sequence, "V" should be understood to designate any nucleotide from G, A, or C, and "N" should be understood to designate any nucleotide at that position in the sequence. For example, "N" can be G, A, T, C, or U. Note that the "R" at position -1 and the "G" at position +3 (the "A" of ATG is position 0) are considered the most critical positions in terms of Kozak strength. However, because the presence of a "G" at position +3 in a transgene sequence depends on the ORF being encoded, the +3 position is not considered herein to be part of the "core" Kozak sequence.

[0183] The bases found in the first six positions of a complete Kozak sequence are varied (noted above as (gcc)gcc) such that any base can be found at those positions. Thus, a complete Kozak consensus sequence can be considered to contain a "core" Kozak sequence consisting of a portion of the complete Kozak sequence with reduced variability, notated above as RccAUG. The "core" Kozak consensus sequence is defined herein as RVVAUG for mRNA and RVVATG for DNA, where "R" should be understood to designate a purine (i.e., A or G) at that position in the sequence and "V" should be understood to designate any nucleotide from G, A, or C.

[0184] In one preferred embodiment of the present invention, the Kozak sequence comprises the sequence RVVATG (SEQ ID NO: 28); where "R" should be understood to designate a purine (i.e., A or G) at that position in the sequence, and "V" should be understood to designate any nucleotide from G, A, or C.

[0185] In one embodiment of the present invention, the Kozak sequence comprises the sequence RNNATG (SEQ ID NO: 125); wherein "R" should be understood to designate a purine (i.e., A or G) at that position in the sequence, and "N" should be understood to designate any nucleotide from G, A, T / U or C, recognizing that use of "T / U" may result in reduced levels of expression in the absence of TRAP.

[0186] In some embodiments, the Kozak sequence overlaps with the 3'-terminal KAGNN repeat of or a portion of the TRAP binding site. Thus, a core Kozak sequence can overlap with the 3'-terminal KAGNN repeat of or a portion of the TRAP binding site.

[0187] A summary of the preferred overlapping tbs and Kozak consensus sequences is provided in Figure 2C.

[0188] In a preferred embodiment, the 3' terminal KAGNN repeat of or part of the TRAP binding site overlaps with at least the first, or first two nucleotides of the ATG triplet within the Koa-Kozak sequence.

[0189] As described herein, in one aspect of the present invention, the 3'-terminal KAGNN repeat of or a portion of the TRAP binding site overlaps with the ATG start codon of the nucleotide of interest (transgene ORF). In one aspect, the 3'-terminal KAGNN repeat of or a portion of the TRAP binding site overlaps with the first one or two ATG start codons of the nucleotide of interest (transgene ORF).

[0190] In one aspect, the overlapping tbs-Kozak sequences can have the consensus sequence KAGNNG (SEQ ID NO: 113), where "NN" are the first two nucleotides within the ATG triplet of the Kozak sequence.

[0191] The consensus sequence may be KAGATG (SEQ ID NO: 114); where "K" is either G or T / U.

[0192] In one aspect, the overlapping tbs-Kozak sequence can be GAGATG (SEQ ID NO: 29) as shown in Figure 2C.

[0193] In one embodiment, the 3' terminal KAGNN repeat of or part of the TRAP binding site overlaps the first nucleotide of the ATG triplet within the nucleotide of interest.

[0194] In one aspect, the sequence can include the sequence KAGNNTG (SEQ ID NO: 115), where the second "N" is the first nucleotide in the ATG triplet. The consensus sequence can be KAGNATG (SEQ ID NO: 116); where "K" should be understood to designate G or T / U at that position in the sequence, and "N" should be understood to designate any nucleotide from G, A, T, U, or C, but preferably "V," i.e., G, A, or C. For example, the overlapping sequence can be KAGVATG (SEQ ID NO: 30), as shown in Figure 2C.

[0195] In one embodiment, the overlapping Kozak sequence and TRAP binding site or portion thereof for use in the nucleic acids of the invention has the following sequence: (a) GAGATG (SEQ ID NO: 29); (b) KAGVATG (SEQ ID NO: 30); (c) KAGVVATG (SEQ ID NO: 31); (d) KAGRVVATG (SEQ ID NO: 32); or (e) KAGNRVVATG (SEQ ID NO: 33); including one of the following: where "K" can be T or G, "R" should be understood to designate a purine (i.e., A or G) at that position in the sequence, "V" should be understood to designate any nucleotide from G, A, or C, and "N" should be understood to designate any nucleotide at that position in the sequence. For example, "N" can be G, A, T, C, or U.

[0196] In one embodiment, the nucleic acid sequence of the invention has the following sequence: (a) GAGATG (SEQ ID NO: 29) or KAGATG (SEQ ID NO: 114); (b) KAGVATG (SEQ ID NO: 30); (c) KAGVVATG (SEQ ID NO: 31); (d) KAGRVVATG (SEQ ID NO: 32); or (e) KAGNRVVATG (SEQ ID NO: 33); including one of the following: where "K" can be T or G, "R" should be understood to designate a purine (i.e., A or G) at that position in the sequence, "V" should be understood to designate any nucleotide from G, A, or C, and "N" should be understood to designate any nucleotide at that position in the sequence. For example, "N" can be G, A, T, C, or U.

[0197] Preferred overlapping tbs or portions thereof and core Kozak sequences corresponding to the consensus sequences GAGATG (SEQ ID NO:29), KAGVATG (SEQ ID NO:30) and KAGVVATG (SEQ ID NO:31) for use in the nucleic acids of the invention include the following (based on the consensus tbs repeat sequence of KAGNN defined herein and the consensus "core" Kozak sequence RVVATG defined herein):

[0198] (a) GAGATG (SEQ ID NO: 29); (b) GAGAATG (SEQ ID NO: 69); (c) GAGCATG (SEQ ID NO: 70); (d) GAGGATG (SEQ ID NO: 71); (e) TAGAATG (SEQ ID NO: 72); (f) TAGCATG (SEQ ID NO: 73); (g) TAGGATG (SEQ ID NO: 74); (h) GAGAAATG (SEQ ID NO: 75); (i) GAGACATG (SEQ ID NO: 76); (j) GAGAGATG (SEQ ID NO: 77); (k) GAGCAATG (SEQ ID NO: 78); (l) GAGCCATG (SEQ ID NO: 79); (m) GAGCGATG (SEQ ID NO: 80); (n) GAGGAATG (SEQ ID NO: 81); (o) GAGGCATG (SEQ ID NO: 82); (p)GAGGGATG (SEQ ID NO: 83); (q) TAGAAATG (SEQ ID NO: 84); (r) TAGACATG (SEQ ID NO: 85); (s) TAGAGATG (SEQ ID NO: 86); (t) TAGCAATG (SEQ ID NO: 87); (u) TAGCCATG (SEQ ID NO: 88); (v) TAGCGATG (SEQ ID NO: 89); (w) TAGGAATG (SEQ ID NO: 90); (x) TAGGCATG (SEQ ID NO: 91); (y) TAGGGATG (sequence number 92).

[0199] In some embodiments, the nucleic acid sequence is the following sequence: (a) KAGCCGAGATG (SEQ ID NO: 34); (b) KAGNGGAGCCATG (SEQ ID NO: 35); or (c) KAGNNGAGACCATG (SEQ ID NO: 36); (d) KAGGCGAGCATG (SEQ ID NO: 37); including one of the following: Here, "K" can be T or G, and "N" should be understood to designate any nucleotide at that position in the sequence. For example, it can be G, A, T, C, or U.

[0200] Preferably, the nucleic acid sequence has the following sequence: (a) KAGCCGAGATG (SEQ ID NO: 34); or (b) KAGNGGAGCCATG (SEQ ID NO: 35); including one of the following: Here, "K" can be T or G, and "N" should be understood to designate any nucleotide at that position in the sequence. For example, it can be G, A, T, C, or U.

[0201] In a preferred embodiment, the nucleic acid sequence of the present invention comprises overlapping tbs and Kozak sequences: GAGTTTAGCGGAGTGGAGAAGAGCGGAGCCGAGCCTAGCAGAGACGAGCCGAGATG (SEQ ID NO: 60).

[0202] Suppression or prevention of translation of an NOI should be understood as a change in the amount of product (e.g. protein) of the NOI that is translated during viral vector production compared to the amount expressed in the absence of the nucleic acid sequence of the invention at a comparable time point. Such a change in translation results in a suppression or prevention of expression of the protein encoded by the NOI.

[0203] In one embodiment, the nucleic acid sequence of the present invention is capable of interacting with TRAP such that translation of the nucleotide of interest is suppressed or prevented in the viral vector producing cell.

[0204] Translation of the NOI at any given time during vector construction may be reduced to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the amount translated in the absence of the nucleic acid sequence of the present invention at the same time during vector construction.

[0205] Translation of the NOI at any given time during vector construction may be reduced to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the amount translated in the absence of the nucleic acid sequence of the present invention at the same time during vector construction.

[0206] In the context of the present invention, translation of the NOI at any given time during vector construction may be reduced to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the amount translated in the presence of a nucleic acid sequence comprising a non-overlapping Kozak and tbs sequence (unlike the nucleic acid sequence of the present invention) at the same time during vector construction.

[0207] In the context of the present invention, translation of the NOI at any given time during vector construction may be reduced to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the amount translated in the presence of a nucleic acid sequence comprising a non-overlapping Kozak and tbs sequence (unlike the nucleic acid sequence of the present invention) at the same time during vector construction.

[0208] Preventing translation of an NOI should be understood as reducing the amount of translation to essentially zero.

[0209] Protein expression from the NOI at any given time during vector construction may be reduced to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the amount expressed in the absence of the nucleic acid sequence of the present invention at the same time during vector construction.

[0210] Protein expression from the NOI at any given time during vector construction may be reduced to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the amount expressed in the absence of the nucleic acid sequence of the invention at the same time during vector construction.

[0211] In the context of the present invention, expression of protein from an NOI at any given time during vector construction may be reduced to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the amount expressed in the presence of a nucleic acid sequence comprising non-overlapping Kozak and tbs sequences (different from the nucleic acid sequence of the present invention) at the same time during vector construction.

[0212] In the context of the present invention, expression of protein from an NOI at any given time during vector construction may be reduced to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the amount expressed in the presence of a nucleic acid sequence comprising non-overlapping Kozak and tbs sequences (unlike the nucleic acid sequence of the present invention) at the same time during vector construction.

[0213] Preventing expression of a protein from an NOI should be understood as reducing the amount of protein expressed to essentially zero.

[0214] Methods for analysing and / or quantitating the translation of NOIs are well known in the art.

[0215] Protein products from lysed cells can be analyzed using methods such as SDS-PAGE analysis with visualization by Coomassie or silver staining. Alternatively, protein products can be analyzed using Western blotting or enzyme-linked immunosorbent assay (ELISA) using an antibody probe that binds the protein product. Protein products in intact cells can be analyzed by immunofluorescence.

[0216] Improved Leader Sequence When applying the TRAP system to different promoters (containing different native 5'UTRs of different lengths and compositions), it would be desirable to simply apply the tbs sequence within the promoter-UTR context to confer efficient repression by TRAP while also maintaining good levels of expression without TRAP. From the start of this study, it was unknown what the achievable levels of repression mediated by TRAP-tbs would be when tbs was inserted into the native UTR of various constitutive promoters. Ideally, when modifying a selected promoter, it would be advantageous to be able to provide a single, conserved 5'UTR leader sequence along with the tbs to avoid any potential variability in repression levels that may be induced by the native 5'UTR sequence. Surprisingly, it was found that the first exon of the EF1α promoter (SEQ ID NO: 25) consistently provided good levels of transgene repression by TRAP compared to a 5'UTR leader containing the native leader sequence, and this leader also provided good levels of transgene expression in the absence of TRAP.

[0217] In some embodiments, the nucleic acid sequence comprises a 5' leader sequence upstream of the tbs or a portion thereof. The leader sequence can be immediately upstream of the TRAP binding site or a portion thereof, i.e., there can be no additional sequence separating the leader sequence from the TRAP binding site or a portion thereof. If the 5' leader is derived from a splicing event, the sequence from the exon / exon junction to the tbs should be kept to a minimum length (preferably ≦12 nt). The leader sequence can comprise a sequence derived from the non-coding EF1α exon 1 region. In preferred embodiments, the leader sequence comprises the sequence defined in SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:93.

[0218] multiple cloning site To improve the tractability of the TRIP system, it would be desirable to have the ability to directly clone an NOI into an expression cassette containing a promoter-5'UTR-tbs sequence via several different restriction enzyme (RE) options, i.e., to incorporate a multiple cloning site (MCS) between the tbs and Kozak sequence (see Figure 2A). We have demonstrated that several different MCSs can be tolerated by the TRIP system, i.e., transgene suppression can still be obtained when an MCS is used. This was unexpected, given that the 5'UTR leader sequence can modulate the degree of TRAP-mediated suppression, that proximity of the tbs to the ATG start codon is important, and that an efficient Kozak sequence must be maintained with or between the MCS and NOI start codons to ensure efficient translation initiation. Additionally, the number and / or combination of RE sites that could be used while maintaining TRAP-mediated suppression could not be predicted.

[0219] This required a "compression" of the sequence so that several (overlapping) RE sites could be incorporated with the shortest possible distance from the tbs to the ATG start codon (to preserve the proximity of the tbs to the ATG), while also maintaining the efficient core Kozak sequence of RVVATG.

[0220] In a further aspect, the present invention provides a nucleic acid sequence comprising a nucleotide of interest, a tbs or portion thereof as described herein, a multiple cloning site (MCS), and a Kozak sequence as described herein, wherein the MCS is located downstream of the tbs or portion thereof and upstream of the Kozak sequence. Suitably, the tbs or portion thereof and the Kozak sequence do not overlap.

[0221] As used herein, a "multiple cloning site" should be understood as a DNA region containing several restriction enzyme recognition sites (restriction enzyme sites) in close proximity to each other. In one embodiment, the RE sites may overlap in an MCS for use in the present invention.

[0222] As used herein, a "restriction enzyme site" or "restriction enzyme recognition site" is a location on a DNA molecule containing a specific sequence of nucleotides, 4 to 8 nucleotides in length, that is recognized by a restriction enzyme. A restriction enzyme recognizes a specific RE site (i.e., a specific sequence) and cuts the DNA molecule within or near the RE site.

[0223] A consensus TRAP binding site sequence capable of binding TRAP is repeated multiple times (e.g., 6, 7, 8, 9, 10, 11, 12 or more times) [KAGNN], where K can be T or G in DNA and U or G in RNA. In one embodiment of the invention, the TRAP binding site or a portion thereof has the sequence KAGN ≧2 (e.g., KAGN 2~3 ) for the avoidance of doubt, this tbs or portion thereof includes, for example, any of the following repeat sequences: UAGNN, GAGNN, TAGNN, UAGNNN, GAGNNN, or TAGNNN. "N" should be understood to designate any nucleotide at that position in the sequence. For example, it can be G, A, T, C, or U. The number of such nucleotides is preferably 2, but can be up to 3, e.g., 1, 2, or 3; the KAG repeats of the 11x repeat tbs or portion thereof can be separated by 3 spacing nucleotides and still retain some TRAP binding activity that results in translational repression. Preferably, no more than one N3 spacer will be used in the 11x repeat tbs or portion thereof to retain maximal TRAP binding activity that results in translational repression.

[0224] In one embodiment, the nucleic acid sequence is the following sequence: (a) GAGCTCTAGAVVATG (SEQ ID NO: 45); (b) GAGCTCGTCGACVATG (SEQ ID NO: 46); (c) GAGCTCGAATTCGAAVVATG (SEQ ID NO: 47); (d) GAGCTCTAGACGTCGACVATG (SEQ ID NO: 48); (e) GAGCTCTAGAATTCGAAVVATG (SEQ ID NO: 49); (f) GAGCTCTAGATATCGATRVVATG (SEQ ID NO: 50); (g) KAGACTAGTACTTAAGCTTRVVATG (SEQ ID NO: 51); (h) GAGCTCTAGACCATG (SEQ ID NO: 52); (i) GAGCTCGTCGACCATG (SEQ ID NO: 53); (j) GAGCTCGAATTCGAACCATG (SEQ ID NO: 54); (k) GAGCTCTAGACGTCGACCATG (SEQ ID NO: 55); (l) GAGCTCTAGAATTCGAACCATG (SEQ ID NO: 56); (m) GAGCTCTAGATATCGATACCATG (SEQ ID NO: 57); or (n) KAGACTAGTACTTAAGCTTACCATG (SEQ ID NO: 58); including one of the following: Here, "K" can be T or G, "R" should be understood to designate a purine (i.e., A or G) at that position in the sequence, and "V" should be understood to designate any nucleotide from G, A, or C.

[0225] In one embodiment, the nucleic acid sequence is the following sequence: (a) GAGCTCTAGACCATG (SEQ ID NO: 52); (b) GAGCTCGTCGACCATG (SEQ ID NO: 53); (c) GAGCTCGAATTCGAACCATG (SEQ ID NO: 54); (d) GAGCTCTAGACGTCGACCATG (SEQ ID NO: 55); (e) GAGCTCTAGAATTCGAACCATG (SEQ ID NO: 56); (f) GAGCTCTAGATATCGATACCATG (SEQ ID NO: 57); or (g) KAGACTAGTACTTAAGCTTACCATG (SEQ ID NO: 58); including one of the following: Here, "K" can be T or G.

[0226] In one embodiment, the nucleic acid sequence is the following sequence: (a) GAGCTCTAGACCATG (SEQ ID NO: 52); (b) GAGCTCTAGACGTCGACCATG (SEQ ID NO: 55); or (c) KAGACTAGTACTTAAGCTTACCATG (SEQ ID NO: 58); including one of the following: Here, "K" can be T or G.

[0227] In a preferred embodiment, the nucleic acid sequence of the invention comprises the overlapping tbs-MCS-Kozak sequence: GAGTTTAGCGGAGTGGAGAAGAGCGGAGCCGAGCCTAGCAGAGACGAGAAGAGCTCTAGACCATG (SEQ ID NO: 61) Includes.

[0228] In one aspect the NOI is operably linked to the tbs or part thereof.

[0229] In one embodiment the tbs or part thereof is capable of interacting with TRAP such that translation of the NOI is repressed in viral vector producing cells.

[0230] In one embodiment the NOI is translated in a target cell that lacks TRAP.

[0231] In one embodiment, tbs or a portion thereof is selected from the group consisting of the sequence KAGN 2~3 Contains multiple repetitions of

[0232] In one embodiment, the tbs or a portion thereof comprises multiple repeats of the sequence KAGN2.

[0233] In one embodiment, the tbs or a portion thereof comprises at least six repeats of the sequence KAGN2. For example, the tbs or a portion thereof may comprise any one of SEQ ID NOs: 8-19 or 22.

[0234] In one embodiment, tbs or a portion thereof is selected from the group consisting of the sequence KAGN 2~3 For example, the tbs or a portion thereof may include any one of SEQ ID NOs: 8, 9, 14-17, 20-24. Preferably, the number of KAGNNN repeats may be 1 or less. For example, the tbs or a portion thereof may include any one of SEQ ID NOs: 8, 9, 14-17, 19-24.

[0235] In one embodiment, the tbs or a portion thereof comprises at least 8 to 11 repeats of the sequence KAGN2.

[0236] In one embodiment, tbs or a portion thereof is selected from the group consisting of the sequence KAGN 2~3 Suitably the number of KAGNNN repeats is 3 or fewer. For example, the tbs or a portion thereof may comprise any one of SEQ ID NOs: 8, 9, 14, 20-22.

[0237] In one embodiment, the Kozak sequence comprises the sequence RVVATG (SEQ ID NO: 28); where "R" should be understood to designate a purine (i.e., A or G) at that position in the sequence, and "V" should be understood to designate any nucleotide from G, A, or C.

[0238] In one embodiment, the Kozak sequence comprises the sequence RNNATG (SEQ ID NO: 125); where "R" should be understood to designate a purine (i.e., A or G) at that position in the sequence, and "N" should be understood to designate any nucleotide from G, A, T / U, or C.

[0239] In one embodiment, the distance from the end of the transcription start site / promoter to the start of the tbs or part thereof is less than 34 nucleotides.

[0240] In one embodiment, the distance from the end of the transcription start site / promoter to the start of the tbs or part thereof is less than 13 nucleotides.

[0241] In one embodiment, the tbs or portion thereof lacks a type II restriction enzyme site, preferably a SapI restriction enzyme site.

[0242] In one embodiment, the nucleic acid sequence comprises a 5' leader sequence upstream of the tbs or portion thereof. The leader sequence may be immediately upstream of the TRAP binding site or portion thereof, i.e., there may be no additional sequence separating the leader sequence from the TRAP binding site or portion thereof.

[0243] In one embodiment, the leader sequence comprises a sequence derived from the non-coding EF1α exon 1 region.

[0244] In one embodiment, the leader sequence comprises the sequence defined in SEQ ID NO:25 or SEQ ID NO:26.

[0245] In one embodiment, the nucleic acid sequence comprises an IRES.

[0246] In one embodiment, the nucleic acid sequence comprises a spacer sequence between the IRES and the tbs or a portion thereof.

[0247] In one embodiment, the spacer is 0 to 30 nucleotides in length.

[0248] In one embodiment, the spacer is 15 nucleotides in length.

[0249] In one embodiment the spacer is 3 or 9 nucleotides from the 3' end of the tbs or portion thereof and downstream start codon of the NOI.

[0250] In one embodiment, the spacer comprises the sequence defined in any one of SEQ ID NOs: 38 to 44, and preferably the spacer comprises the sequence defined in SEQ ID NO: 38.

[0251] In one aspect the NOI produces a therapeutic effect.

[0252] In one embodiment, the nucleic acid sequence further comprises an RRE sequence or a functional substitute thereof.

[0253] In one embodiment, the nucleic acid sequence is a vector transgene expression cassette.

[0254] In one embodiment, the nucleic acid sequence of the present invention further comprises a promoter.Typically, the transcription of promoter results in the 5'UTR encoded in the resulting mRNA transcript.Promoter can be any promoter known in the art and suitable for controlling the expression of nucleotide of interest.For example, promoter can be EF1α, EFS, CMV or CAG.

[0255] In preferred embodiments, the overlapping tbs and Kozak sequences described herein are located within the 5'UTR of a promoter, wherein the 5'UTR may comprise native sequences from the relevant promoter, or more preferably, the 5'UTR is composed of the 5'UTR sequences described herein.

[0256] In a preferred embodiment, a sequence comprising the compressed / overlapping MCS between the tbs and Kozak sequences described herein is located within said 5'UTR.

[0257] A sequence comprising an overlapping tbs and a Kozak sequence as described herein or a compressed / overlapping MCS between a tbs and a Kozak sequence as described herein may be located at the 3' end of the 5'UTR.

[0258] Preferably, the 5'UTR comprises one of the following sequences: SEQ ID NOs: 29 to 37, 45 to 58, 69 to 92, and 108 to 116. More preferably, the 5'UTR comprises SEQ ID NO: 29 or SEQ ID NO: 108. Even more preferably, the 5'UTR comprises SEQ ID NO: 29.

[0259] The promoter-5'UTR region may contain an intron. The intron may be a natural intron or a heterologous intron. For example, the promoter may be EF1α or CAG.

[0260] The promoter may be a promoter typically used in an intronless viral vector genome, such as CMV.

[0261] In a preferred embodiment, promoter-5'UTR region is engineered to contain an artificial 5'UTR that contains a heterologous intron.Therefore, promoter-5'UTR region is engineered to contain a heterologous exon-intron-exon sequence, and the mature 5'UTR encoded in mRNA transcript is generated by the excision of intron by splicing.Promoter-5'UTR sequence can be engineered using methods known in the art.For example, promoter can be engineered as described herein (see Example 8).

[0262] Preferably, the expression of the transgene protein from its mature mRNA resulting from the splicing out of the intron or heterologous intron is efficiently suppressed by TRAP. Suitably, the intron or heterologous intron may be the EF1α intron sequence as set forth in SEQ ID NO: 122.

[0263] The intron or heterologous intron can be located upstream, i.e., 5', of the overlapping tbs and Kozak sequence described herein or of the sequence comprising the MCS between the tbs and Kozak sequence described herein.

[0264] The 5'UTR has the following sequence (chicken β-actin / rabbit β-globin chimeric 5'UTR-intron, exon sequences in bold (spliced ​​together to form the 5'UTR leader)): may include:

[0265] The 5'UTR has the following sequence (EF1a 5'UTR-intron, exon sequence in bold (spliced ​​together to become the 5'UTR leader)): CTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGTGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGTGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCAGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGCCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCACAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCCAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAAAA(SEQ ID NO: 122) may include:

[0266] In one embodiment, the promoter has the sequence (exon sequences in bold (spliced ​​together to become the 5'UTR leader), tbs consensus in italics): 2-3 ] 10-11 (SEQ ID NO: 123) Includes.

[0267] In one embodiment, the promoter has the sequence (exon sequences in bold (spliced ​​together to become the 5'UTR leader), tbs consensus in italics): CTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGTGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGTGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCAGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGCCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCACAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCCAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAAAA[KAGN 2-3] 10-11 (SEQ ID NO: 124) Includes.

[0268] In one embodiment, the promoter has the sequence (chicken β-actin / rabbit β-globin chimeric 5′UTR-intron with tbs-kzkV0.G variant, exon sequences in bold (spliced ​​together to become 5′UTR leader), tbskzkV0.G in italics): Includes.

[0269] In one embodiment, the promoter has the sequence (EF1a 5'UTR-intron with overlapping tbs and Kozak sequences (tbskzkV0.G variant), exon sequences in bold (spliced ​​together to become the 5'UTR leader), tbskzkV0.G in italics): Includes.

[0270] Spliced ​​sequence corresponding to SEQ ID NO: 117; 5'UTR leader sequence in bold, tbskzkV0.G in italics: CGGCGGGCGGGAACGTTGCCTTCGCCCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGCTCCTGGGCAAAGAGTTTAGCGGAGTGGAGAAGAGCGGAGCCGAGCCTAGCAGAGACGAGCCGAGATG (SEQ ID NO: 119) Spliced ​​sequence corresponding to SEQ ID NO: 118; 5'UTR leader sequence in bold, tbskzkV0.G in italics: CTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTGTCGTGAAAAGAGTTTAGCGGAGTGGAGAAGAGCGGAGCCGAGCCTAGCAGAGACGAGCCGAGATG (SEQ ID NO: 120) Exemplary Nucleic Acid Sequences Exemplary nucleic acid sequences of the invention are shown below.

[0271] SEQ ID NO: 62-L33 Improved leader, exemplary nucleic acid sequence 1 containing optimal (overlapping) tbs([KAGNN]8)-Kozak junction CTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGAGTTTAGCGGAGTGGAGAAGAGCGGAGCCGAGCCGAGATG Sequence number 63-L33 Improved reader, optimal (overlapping) tbs ([KAGNN] 11 Exemplary nucleic acid sequences containing 2-Kozak junctions CTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGAGTTTAGCGGAGTGGAGAAGAGCGGAGCCGAGCCTAGCAGAGACGAGCCGAGATG Sequence number 64-L12 improved reader, optimal (overlapping) tbs ([KAGNN] 11 )-Kozak-containing exemplary nucleic acid sequence 3 CTTTTTCGCAACGAGTTTAGCGGAGTGGAGAAGAGCGGAGCCGAGCCTAGCAGAGACGAGCCGAGATG SEQ ID NO: 65—Exemplary nucleic acid sequence for an intron-containing 5′UTR 4, spliced ​​leader including L33, optimal (overlapping) tbs ([KAGNN] 11 )-resulting in Kozak junction CTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTGTCGTGAAAAGAGTTTAGCGGAGTGGAGAAGAGCGGAGCCGAGCCTAGCAGAGACGAGCCGAGATG SEQ ID NO: 66 - Improved spacer, exemplary nucleic acid sequence 5 containing optimal (overlapping) tbs([KAGNN]8)-Kozak junctions ATAGCAGAGACGGCTGAGTTTAGCGGAGTGGAGAAGAGCGGAGCCGAGCCGAGATG Sequence number 67-L33 Improved reader tbs([KAGNN] 11 )- MCS - Exemplary nucleic acid sequence containing Kozak 6 CTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGAGTTTAGCGGAGTGGAGAAGAGCGGAGCCGAGCCTAGCAGAGACGAGAA GAGCTCTAGA CCATG SEQ ID NO: 68 - Improved spacer, tbs([KAGNN] 11 )- MCS - Exemplary nucleic acid sequences containing Kozak 7 ATAGCAGAGACGGCTGAGTTTAGCGGAGTGGAGAAGAGCGGAGCCGAGCCTAGCAGAGACGAGAA GAGCTCTAGA CCATG In one embodiment, the nucleic acid sequence comprises any one of SEQ ID NOs: 62-68.

[0272] In one embodiment, the nucleic acid sequence is (a)(i) SEQ ID NO: 25 or 26; and / or (ii) any one of SEQ ID NOs: 38-44; (b) any one of SEQ ID NOs: 10 to 13, 15 to 19, 23, and 24; and (c)(i) any one of SEQ ID NOs: 29-36, preferably any one of SEQ ID NOs: 34-36; or (ii) any one of SEQ ID NOs: 45 to 58, preferably any one of SEQ ID NOs: 52 to 58 Includes.

[0273] In one embodiment, the nucleic acid sequence is (a) SEQ ID NO: 25 or 26; (b) any one of SEQ ID NOs: 10-13, 16-19, 23, and 24; and (c) any one of SEQ ID NOs: 29 to 36, preferably any one of SEQ ID NOs: 34 to 36 Includes.

[0274] In one embodiment, the nucleic acid sequence is (a) any one of SEQ ID NOs: 38-44; (b) any one of SEQ ID NOs: 10-13, 16-19, 23, and 24; and (c) any one of SEQ ID NOs: 29 to 36, preferably any one of SEQ ID NOs: 34 to 36 Includes.

[0275] In one embodiment, the nucleic acid sequence is (a) SEQ ID NO: 25 or 26; (b) any one of SEQ ID NOs: 10-13, 15-19, 23, and 24; and (c) any one of SEQ ID NOs: 45 to 58, preferably any one of SEQ ID NOs: 52 to 58 Includes.

[0276] In one embodiment, the nucleic acid sequence is (a) any one of SEQ ID NOs: 38-44; (b) any one of SEQ ID NOs: 10-13, 15-19, 23, and 24; and (c) any one of SEQ ID NOs: 45 to 58, preferably any one of SEQ ID NOs: 52 to 58 Includes.

[0277] Nucleotide of interest In one embodiment of the invention, the nucleotide of interest is translated in a target cell that lacks TRAP.

[0278] A "target cell" should be understood as a cell in which it is desired to express the NOI. The NOI may be introduced into the target cell using a viral vector of the present invention. Delivery to the target cell may be performed in vivo, ex vivo or in vitro.

[0279] In a preferred embodiment, the nucleotide of interest produces a therapeutic effect.

[0280] 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, intracellular localization signals, tumor suppressor proteins, growth factors, membrane proteins, receptors, vasoactive proteins and peptides, antiviral proteins and ribozymes, and derivatives thereof (such as 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.

[0281] In one aspect, the NOI may be useful in the treatment of neurodegenerative disorders.

[0282] In another embodiment, the NOI may be useful in the treatment of Parkinson's disease.

[0283] 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).

[0284] In another embodiment, the NOI may encode vesicular monoamine transporter 2 (VMAT2). In an alternative 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.

[0285] In another embodiment the NOI may encode a therapeutic protein or a combination of therapeutic proteins.

[0286] 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-1β (IL-1β), tumor 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.

[0287] 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-α, interferon-inducible protein, globbeta and tuberculosis-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 Patent Nos. 5,952,199 and 6,100,071, and anti-VEGF receptor antibodies.

[0288] 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 inhibitors, IL1β inhibitors, TGFβ inhibitors, IL-6 inhibitors, IL-23 inhibitors, IL-18 inhibitors, tumour necrosis factor alpha and beta, lymphotoxin alpha and beta, LIGHT inhibitors, alpha synuclein inhibitors, tau inhibitors, beta amyloid inhibitors, IL-17 inhibitors.

[0289] In another embodiment the NOI may encode the cystic fibrosis transmembrane conductance regulator (CFTR).

[0290] In another embodiment the NOI may encode a protein that is normally expressed in ocular cells.

[0291] In another embodiment the NOI may encode a protein that is normally expressed in photoreceptor cells and / or retinal pigment epithelial cells.

[0292] In another embodiment, the NOI may encode a protein selected from the group comprising RPE65, aryl hydrocarbon interacting receptor protein-like 1 (AIPL1), CRB1, lecithin retinal acetyltransferase (LRAT), photoreceptor-specific homeobox (CRX), retinal guanylate cyclise (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, CEP290, RPGR, RS1, RP1, PRELP, glutathione pathway enzymes and opticin.

[0293] In other embodiments the NOI may encode human coagulation factor VIII or IX.

[0294] 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 ketoacid 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 beta, cystinosin, glucosamine The gene may encode one or more proteins involved in metabolism selected from the group including (N-acetyl)-6-sulfatase, N-acetyl-α-glucosaminidase, N-sulfoglucosamine sulfohydrolase, galactosamine-6 ​​sulfatase, arylsulfatase A, cytochrome B-245β, ABCD1, ornithine carbamoyltransferase, argininosuccinate synthase, argininosuccinate lysase, arginase 1, alanine glycoxhylate aminotransferase, ATP-binding cassette, and subfamily B members.

[0295] 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, 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, vesicular endothelial growth factor receptor 2 (VEGFR2).

[0296] In other embodiments the NOI may encode a chimeric antigen receptor (CAR) against an NKG2D ligand selected from the group comprising ULBP1, 2 and 3, H60, Rae-1a, b, g, d, MICA, MICB.

[0297] In a further aspect 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, protease, ribosomal protein (RI) and ribosomal protein (RI). The enzymes may encode toporphyrinogen (PROTO) oxidase, ferrochelatase, α-L-iduronidase, iduronate sulfatase, heparan sulfamidase, N-acetylglucosaminidase, heparan-α-glucosaminide N-acetyltransferase, 3N-acetylglucosamine 6-sulfatase, galactose-6-sulfate sulfatase, β-galactosidase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, and hyaluronidase.

[0298] In addition to the NOI, the vector may also contain or encode an siRNA, shRNA, or regulated shRNA. (Dickins et al. (2005) Nature Genetics 37:1289-1295, Silva et al. (2005) Nature Genetics 37:1281-1288) Indications The vectors, including retroviral vectors and AAV vectors, according to the present invention can 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 nucleotide of interest can be DNA or RNA. Examples of such diseases are as follows: cytokine and cell proliferation / differentiation activity; immunosuppressive or immunostimulatory activity (e.g., for treating immune deficiencies, including infection with human immunodeficiency virus, modulating lymphocyte proliferation; treating cancer and many autoimmune diseases, and preventing transplant rejection or inducing tumor immunity); modulation of hematopoiesis (e.g., treating myeloid or lymphoid diseases); promoting the 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., to recruit specific cell types to sites of injury or infection); hemostatic and thrombolytic activity (e.g., to treat hemophilia and stroke); anti-inflammatory activity (e.g., to treat septic shock or Crohn's disease); macrophage inhibitory activity 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 associated with inflammation); inhibition of the ability of macrophages and T cells to adhere to extracellular matrix components and fibronectin, and disorders responsive to upregulated fas receptor expression in T cells.

[0299] Malignant disorders including cancer, leukemia, benign and malignant tumor growth, invasion and spread, angiogenesis, metastasis, ascites and malignant pleural effusion.

[0300] Arthritis, including rheumatoid arthritis, hypersensitivity, allergic reactions, asthma, autoimmune diseases, including systemic lupus erythematosus, collagen disorders and other diseases.

[0301] 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.

[0302] Diseases of the gastrointestinal tract, including peptic ulcers, ulcerative colitis, Crohn's disease and other diseases.

[0303] Liver disease, including liver fibrosis and cirrhosis.

[0304] Inherited metabolic disorders including phenylketonuria (PKU), Wilson's disease, organic acidemias, urea cycle disorders, cholestasis, and other diseases.

[0305] Kidney and urinary diseases, including thyroiditis or other glandular diseases, glomerulonephritis or other diseases.

[0306] Ear, nose and throat disorders, including otitis or other ear, nose and throat diseases, dermatitis or other skin diseases.

[0307] Dental and oral disorders, including periodontal disease, periodontitis, gingivitis or other dental / oral diseases.

[0308] Orchitis or epididymis - Testicular disease including orchitis, infertility, testicular trauma or other testicular disease.

[0309] Gynecological diseases including placental dysfunction, placental insufficiency, habitual miscarriage, eclampsia, pre-eclampsia, endometriosis and other gynecological diseases.

[0310] Leber's congenital amaurosis (LCA) including LCA10, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, retinouveitic inflammation, optic neuritis, glaucoma including open-angle glaucoma and juvenile congenital glaucoma, intraocular inflammation such as retinitis or cystoid macular edema, sympathetic ophthalmia, scleritis, retinitis pigmentosa, age-related macular degeneration (AMD) and macular degeneration including Best's disease, juvenile macular degeneration including Best's vitelliform macular degeneration, Stargardt's disease, Usher syndrome, Doyne's honeycomb retinal dystrophy, Sorby's macular dystrophy, juvenile glaucoma, glaucoma, retinal dystrophy ... Ophthalmological disorders such as age-related 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 (fondus) disease, ocular trauma, ocular inflammation caused by infection, proliferative vitreoretinopathy, acute ischemic optic neuropathy, excessive scarring e.g. after glaucoma filtration surgery, reactions to ocular transplants, corneal graft rejection, and other ocular diseases such as diabetic macular edema, retinal vein occlusion, RLBP1-related retinal dystrophy, total choroidal atrophy, and color vision disorders.

[0311] Parkinson's disease, complications and / or side effects of Parkinson's disease treatment, AIDS-related dementia complex, HIV-associated encephalopathy, Devic's disease, Sydenham's chorea, Alzheimer's disease and other degenerative diseases, symptoms or disorders of the CNS, stroke, post-polio syndrome, psychiatric disorders, myelitis, encephalitis, subacute sclerosing panencephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, Fabry disease, Gaucher disease, cystinosis, Pompe disease, metachromatic leukodystrophy, Wiscott disease t) Neurological and neurodegenerative disorders including Aldrich syndrome, adrenoleukodystrophy, beta thalassemia, sickle cell disease, Guillain-Barré syndrome, Sydenham chorea, myasthenia gravis, pseudotumor cerebri, Down's syndrome, Huntington's disease, CNS compression or CNS trauma or infection of the CNS, muscular atrophies and dystrophies, diseases, conditions or disorders of the central and peripheral nervous system, amyotrophic lateral sclerosis, spinal muscular atrophy, motor neuron diseases including spinal cord and avulsion injuries.

[0312] Other diseases and conditions such as cystic fibrosis, mucopolysaccharidoses including Sanfilippo syndrome A, Sanfilippo syndrome B, Sanfilippo syndrome C, Sanfilippo syndrome D, Hunter syndrome, Hurler-Scheie syndrome, Morquio syndrome, adenosine deaminase severe combined immunodeficiency (ADA-SCID), X-linked severe combined immunodeficiency, 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, infectious diseases, diabetes, complications or side effects of surgery, bone marrow transplantation or other transplant complications and / or side effects, complications and side effects of gene therapy due to, for example, infection by a virus carrier or AIDS, to suppress or inhibit 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 such as corneas, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue.

[0313] siRNA, microRNA and shRNA In certain other embodiments, the NOI comprises microRNA.MicroRNA is a very large group of small RNAs that are naturally produced in organisms, at least some of which regulate the expression of target genes.The founding 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 worm development.Active RNA species are first transcribed as about 70nt precursors, which are transcribed into about 21nt mature forms.Both let-7 and lin-4 are transcribed as hairpin RNA precursors that are processed into their mature forms by Dicer enzyme.

[0314] In addition to the NOI, the vector may also contain or encode an siRNA, shRNA or regulated shRNA (Dickins et al. (2005) Nature Genetics 37:1289-1295, Silva et al. (2005) Nature Genetics 37:1281-1288).

[0315] Posttranscriptional gene silencing (PTGS) mediated by double-stranded RNA (dsRNA) is a conserved cellular defense mechanism for controlling the expression of foreign genes. Random integration of elements such as transposons or viruses is thought to trigger the expression of dsRNA, which activates the sequence-specific degradation of homologous single-stranded mRNA or viral genomic RNA. This silencing effect is known as RNA interference (RNAi) (Ralph et al. (2005) Nature Medicine 11:429-433). The RNAi mechanism involves the processing of long dsRNA into approximately 21-25 nucleotide (nt) double-stranded RNAs. These products are called small interfering RNAs or silencing RNAs (siRNAs), which are sequence-specific mediators of mRNA degradation. It has been found that in differentiated mammalian cells, dsRNA longer than 30bp activates interferon response, leading to the stop of protein synthesis and non-specific mRNA degradation (Stark et al., Annu Rev Biochem 67:227-64(1998)).However, this response can be avoided by using 21nt siRNA duplex (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)), making it possible to analyze gene function in cultured mammalian cells.

[0316] NOIs and polynucleotides The polynucleotide of the present invention can comprise DNA or RNA. The polynucleotide of the present invention can be single-stranded or double-stranded. It will be understood by those skilled in the art that, due to the degeneracy of the genetic code, many different polynucleotides can encode the same polypeptide. Furthermore, it should be understood that those skilled in the art can use routine techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotide of the present invention to reflect the codon usage of any specific host organism in which the polypeptide of the present invention is to be expressed.

[0317] 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.

[0318] Polynucleotides, such as DNA polynucleotides, can be produced recombinantly, synthetically, or by any means available to those of skill in the art. Polynucleotides, such as DNA polynucleotides, can also be cloned by standard techniques.

[0319] Longer polynucleotides will generally be generated using recombinant means, for example, using polymerase chain reaction (PCR) cloning techniques. This involves generating a pair of primers (e.g., about 15-30 nucleotides) that flank the target sequence desired 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 may be designed to contain appropriate restriction enzyme recognition sites so that the amplified DNA can be cloned into an appropriate vector.

[0320] Major splice donor 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 and represent the locations 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 another splice site.

[0321] Splice sites allow the excision of introns present in pre-mRNA transcripts. Typically, the 5' splice boundary is called a "splice donor site" or "5' splice site," and the 3' splice boundary is called a "splice acceptor site" or "3' splice site." Splice sites include, for example, naturally occurring splice sites, engineered or synthetic splice sites, canonical or consensus splice sites, and / or non-canonical splice sites, such as cryptic splice sites.

[0322] 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.

[0323] 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.

[0324] In one aspect, the present invention also provides a nucleic acid sequence according to the invention described herein, wherein the nucleic acid sequence is comprised within the RNA genome of a lentiviral vector, and wherein a major splice donor site within the RNA genome of the lentiviral vector is inactivated, e.g. mutated or deleted.

[0325] In one aspect, the present invention also provides a nucleic acid sequence according to the invention described herein, wherein the nucleic acid sequence is operably linked to the RNA genome of a lentiviral vector, and wherein a major splice donor site within the RNA genome of the lentiviral vector is inactivated, e.g. mutated or deleted.

[0326] In one aspect, a nucleotide sequence is provided that encodes the RNA genome of a lentiviral vector, wherein a major splice donor site in the RNA genome of said lentiviral vector is inactivated, e.g., mutated or deleted.

[0327] The terms "canonical splice site" or "consensus splice site" may be used interchangeably and refer to a splice site that is conserved among species.

[0328] 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 a nearly invariant dinucleotide at each end of the intron: GT at the 5' end of the intron and AG at the 3' end of the intron.

[0329] The canonical splice donor site consensus sequence (for 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). It is well known in the art that splice donors can deviate from this consensus, particularly in viral genomes where other constraints are imposed on the same sequence, such as secondary structures within the vRNA packaging region. Non-canonical splice sites are also well known in the art, but occur less frequently than canonical splice donor consensus sequences.

[0330] "Major splice donor site" refers to the first (dominant) splice donor site in the viral vector genome, encoded and integrated within the natural viral RNA packaging sequence, typically located in the 5' region of the viral vector nucleotide sequence.

[0331] In one aspect, the nucleotide sequence encoding the RNA genome of the lentiviral vector 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.

[0332] The major splice donor site is located in the 5' packaging region of the lentiviral genome.

[0333] In the case of the HIV-1 virus, the major splice donor consensus sequence (in 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).

[0334] In one aspect of the invention, the splice donor region, i.e., the region of the vector genome that contains the major splice donor site before mutation, has the following sequence: GGGGCGGCGACTGGTGAGTACGCCAAAAAT (SEQ ID NO: 94) may have:

[0335] In one aspect of the invention, the mutated splice donor region has the sequence: GGGGCGGCGACTGCAGACAACGCCAAAAAT (SEQ ID NO: 95-MSD-2KO) may include:

[0336] In one aspect of the invention, the mutated splice donor region has the sequence: GGGGCGGCGAGTGGAGACTACGCCAAAAAT (SEQ ID NO: 104-MSD-2KOv2) may include:

[0337] In one aspect of the invention, the mutated splice donor region has the sequence: GGGGAAGGCAACAGATAAATATGCCTTAAAAT (SEQ ID NO: 105-MSD-2KOm5) may include:

[0338] In one aspect of the invention, prior to modification, the splice donor region has the following sequence: GGCGACTGGTGAGTACGCC (SEQ ID NO: 102) may include:

[0339] 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 aspect of the present invention, this sequence (SL2) can be deleted from the nucleotide sequences according to the present invention described herein.

[0340] Thus, the present invention encompasses nucleotide sequences that do not comprise SL2. The present invention encompasses nucleotide sequences that do not comprise a sequence according to SEQ ID NO:102.

[0341] In one aspect of the invention, the major splice donor site may have the following consensus sequence, where R is a purine and " / " is the cleavage site: TG / GTRAGT (SEQ ID NO: 96) In one aspect, R can be guanine (G).

[0342] In one aspect of the invention, the major splice donor and cryptic splice donor regions can have the following core sequences, where " / " is the cleavage site at the major splice donor and cryptic splice donor sites: / GTGA / GTA (SEQ ID NO: 106).

[0343] In one aspect of the invention, an MSD-mutated vector genome can have at least two mutations in the major splice donor and cryptic splice donor "regions" (SEQ ID NO: 106), with the first and second "GT" nucleotides immediately 3' to the major splice donor and cryptic splice donor nucleotides, respectively.

[0344] In one aspect of the invention, the major splice donor consensus sequence is CTGGT (SEQ ID NO: 97). A major splice donor site may contain the sequence CTGGT.

[0345] In one aspect, the nucleotide sequence comprises the sequence set forth in any of SEQ ID NOs: 94, 96, 97, 102, 103 and / or 106 prior to splice site inactivation.

[0346] In one aspect, the nucleotide sequence comprises an inactivated major splice donor site that has a cleavage site between nucleotides that would otherwise correspond to nucleotides 13 and 14 of SEQ ID NO:94.

[0347] In accordance with the invention described herein, the nucleotide sequence also contains an inactive cryptic splice donor site. In one aspect, the nucleotide sequence does not contain an active cryptic splice donor site adjacent to (3' of) 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.

[0348] The term "cryptic splice donor site" refers to a nucleic acid sequence that does not normally function as a splice donor site or that is utilized less efficiently as a splice donor site due to the context of adjacent sequences (e.g., the presence of a nearby "preferred" splice donor), but that can be activated to become a more efficiently functioning splice donor site by mutation of adjacent sequences (e.g., mutation of a nearby "preferred" splice donor).

[0349] In one aspect, the cryptic splice donor site is the first cryptic splice donor site 3' to the major splice donor.

[0350] In one aspect, the cryptic splice donor site is located 3' to the major splice donor site and within 6 nucleotides of the major splice donor site. Preferably, the cryptic splice donor site is located within 4 or 5, preferably 4, nucleotides of the major splice donor cleavage site.

[0351] In one aspect of the invention, the cryptic splice donor site has the consensus sequence TGAGT (SEQ ID NO: 103).

[0352] In one aspect, the nucleotide sequence comprises an inactivated cryptic splice donor site that would otherwise have a cleavage site between nucleotides corresponding to nucleotides 17 and 18 of SEQ ID NO:94.

[0353] In one aspect of the present invention, the major splice donor site and / or the adjacent cryptic splice donor site contain a "GT" motif. In one aspect of the present 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."

[0354] In one aspect, the splice donor region has the following sequence: CAGACA (SEQ ID NO: 98) may include:

[0355] For example, in one aspect, the mutated splice donor region has the following sequence: GGCGACTGCAGACAACGCC (SEQ ID NO: 99) may include:

[0356] A further example of an inactivating mutation is referred to herein as "MSD-2KOv2."

[0357] In one aspect, the mutated splice donor region has the following sequence: GTGGAGACT (SEQ ID NO: 100) may include:

[0358] For example, in one aspect, the mutated splice donor region has the following sequence: GGCGAGTGGAGACTACGCC (SEQ ID NO: 101) may include:

[0359] For example, in one aspect, the mutated splice donor region has the following sequence: AAGGCAACAGATAAATATGCCTT (SEQ ID NO: 107) may include:

[0360] In one aspect, 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."

[0361] 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.

[0362] In one aspect, the mutation is a functional mutation to eliminate or suppress splicing activity in the splice region. The nucleotide sequences described herein can contain mutations or deletions in any of the nucleotides in any of SEQ ID NOs: 94, 96, 97, 102, 103 and / or 106.

[0363] Suitable mutations will be known to those skilled in the art and are described herein.

[0364] For example, point mutations can be introduced into nucleic acid sequences. As used herein, the term "point mutation" refers to any change to a single nucleotide. Point mutations include, for example, deletions, transitions, and transversions, and when present in a protein-coding sequence, they 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 therein.

[0365] 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 cryptic splice donor sites. In one aspect, 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 and " / " is the cleavage site. It is well known in the art that splice donor cleavage sites can deviate from this consensus, especially in viral genomes where other constraints are imposed on the same sequence, such as secondary structures within the vRNA packaging region. 3 T 4 The dinucleotide is generally the least variable sequence within the canonical splice donor consensus sequence, G 3 and or T 4 It is well known that mutations to T are most likely to achieve the greatest attenuation effect. For example, for the major splice donor site in the HIV-1 viral vector genome, this is 1 G 2 / G 3 T 4 where " / " is the cleavage site. For example, for a potential splice donor site in the HIV-1 viral vector genome, this is G 1 A 2 / G 3 T 4 where " / " is the cleavage site. Additionally, point mutation(s) can be introduced adjacent to the splice donor site. For example, point mutations can be introduced upstream or downstream of the splice donor site. In embodiments where a nucleic acid sequence comprising a major splice donor site and / or a cryptic splice donor site is mutated by introducing multiple point mutations therein, point mutations can be introduced upstream and / or downstream of the cryptic splice donor site.

[0366] Construction of splice site mutants Splice site variants for use in the present 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.

[0367] Other known techniques that allow for the alteration of DNA sequences include recombinant approaches such as Gibson assembly, Golden-gate cloning and in-fusion.

[0368] Alternatively, oligonucleotide-directed site-specific (or segment-specific) mutagenesis procedures can be used to provide altered sequences with specific codons altered according to the required substitution, deletion, or insertion. Deletion or truncation derivatives of splice site mutants can also be constructed by utilizing convenient restriction endonuclease sites flanking the desired deletion.

[0369] Following restriction, the overhangs can be filled in and the DNA religated.

[0370] Exemplary methods for making the above modifications are disclosed by Sambrook et al. (Molecular cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, 1989).

[0371] Splice site mutants can also be constructed using techniques of chemical mutagenesis (Drinkwater and Klinedinst, 1986), by PCR mutagenesis, chemical mutagenesis, forced nucleotide misincorporation (e.g., Liao and Wise, 1990), or by the use of randomly mutagenized oligonucleotides (Horwitz et al., 1989).

[0372] The present invention provides a method for producing a lentiviral vector nucleotide sequence, comprising: (i) providing a nucleotide sequence encoding the RNA genome of a lentiviral vector described herein; and mutating the major splice donor site and the cryptic splice donor sites described herein in said nucleotide sequence; Also provided is a method, including:

[0373] Combination with modified U1 Due to their reduced ability to participate in aberrant splicing events both during LV production and in target cells, MSD-mutated lentiviral vectors are preferable to current standard lentiviral vectors for use as gene therapy vectors. However, until the present invention, production of MSD-mutated vectors has relied on the supply of HIV-1 tat protein (first- and second-generation lentiviral vectors) or was less efficient due to the destabilizing effect of mutating the MSD on vector RNA levels (third-generation vectors). For safety reasons, "reintroducing" tat into current third-generation LV systems is not desirable or justified, and as a result, there is currently no solution to the reduced production titers of MSD-mutated vectors intended for clinical use.

[0374] We demonstrate that high-titer production of MSD-mutated third-generation (i.e., Tat-independent) LVs can be achieved by coexpression of modified U1 snRNAs that are directed to bind to the 5' packaging region of vector genomic RNA during production. Surprisingly, we demonstrate that these modified U1 snRNAs can enhance the production titer of MSD-mutated LVs in a manner independent of the presence of a 5' polyA signal in the 5' R region, demonstrating a novel mechanism beyond the use of modified U1 snRNAs to suppress polyadenylation (so-called U1 interference, [Ui]). Surprisingly, we demonstrate that targeting modified U1 snRNAs to critical sequences in the packaging region results in the greatest enhancement of MSD-mutated LV titers. We also disclose novel sequence mutations within the major splice donor region that result in a less pronounced decrease in MSD-mutated LV titer and maximize the enhancement of titer of such MSD-mutated LV variants by modified U1 snRNA.

[0375] The present inventors have surprisingly found that the output titer of lentiviral vectors can be enhanced by co-expressing a non-coding RNA based on U1snRNA that has been modified so that it no longer targets an endogenous sequence (splice donor site) but now targets a sequence within the vRNA molecule. As demonstrated in the Examples, the present inventors show that the relative enhancement of the output titer of lentiviral vectors carrying attenuating mutations in the major splice donor region (containing the major splice donor and cryptic splice donor sites) by the modified U1snRNA is greater than that of standard lentiviral vectors containing an unmutated major splice donor region.

[0376] As demonstrated in the Examples, vector genomes with a wide range of mutations (point mutations, region deletions, and sequence replacements) within the major splice donor region that result in reduced titers can be used in combination with modified U1 snRNA. This approach can involve coexpressing the modified U1 snRNA with other vector components during vector production. The modified U1 snRNA is designed to eliminate binding to the consensus splice donor site by replacing the target sequence within the vector genome vRNA with a heterologous sequence complementary to the target sequence. The present invention describes various application modes and optimal characteristics of modified U1 snRNA, including the target sequence and complementary length, design, and expression modes.

[0377] In one aspect of the invention described herein, the vectors may be used in combination with modified U1 snRNA, as discussed further below.

[0378] Splicing and polyadenylation are essential processes for mRNA maturation, especially in higher eukaryotes, where most protein-coding transcripts contain multiple introns. Elements within the pre-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 formed by 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 well-defined structure consisting of four stem-loops (West, S., 2012, Biochemical Society Transactions, 40:846-849). At its 5' end, U1snRNA contains a short sequence that is broadly complementary to the 5' splice donor site at the exon-intron junction. U1snRNA is involved in splice site selection and spliceosome assembly by base pairing to the 5' splice donor site. A known function of U1snRNA, other than splicing, is in regulating 3'-end mRNA processing, suppressing premature polyadenylation (polyA) at the initial polyA signal.

[0379] Human U1 snRNA (small nuclear RNA) is 164 nt long and has a well-defined structure consisting of four stem-loops (see Figure 1). U1 snRNA, an endogenous noncoding RNA, binds to a consensus 5' 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, together with stem-loop IV, binds to the Sm protein, which is important for U1 snRNA processing. As defined herein, modified U1 snRNA for use in accordance with the present invention has been modified to introduce a heterologous sequence complementary to a target sequence within a vector genome vRNA molecule at the site of the natural splice donor targeting / annealing sequence (see Figure 1).

[0380] As used herein, the terms "modified U1 snRNA," "redirected U1 snRNA," "retargeted U1 snRNA," "repurposed U1 snRNA," and "mutant U1 snRNA" refer to a U1 snRNA that has been modified so that it is no longer complementary to the consensus 5' splice donor site sequence (e.g., 5'-MAGGURR-3') used 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 is no longer complementary to a splice donor site sequence (e.g., 5'-MAGGURR-3'). Instead, the modified U1 snRNA is designed to target or be complementary to a nucleotide sequence with a unique RNA sequence (target site) within the packaging region of a lentiviral vector genome molecule with a mutated MSD, i.e., a sequence unrelated to vRNA splicing. The nucleotide sequence within the packaging region of a lentiviral vector genome molecule with a mutated MSD can be preselected. Therefore, the modified U1 snRNA is a U1 snRNA whose 5' end is complementary to a nucleotide sequence in the packaging region of a lentiviral vector genome molecule having a mutated MSD. As a result, without wishing to be bound by theory, it is believed that 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, thereby stabilizing the vRNA and resulting in an increased output vector titer of the MSD-mutated lentiviral vector.

[0381] 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 in an intron. A natural splice donor annealing sequence can be 5'-ACUUACCUG-3'.

[0382] 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'.

[0383] As used herein, the terms "nucleotide sequence within the packaging region of a lentiviral vector genome sequence having a mutated MSD," "target sequence," and "target site" refer to a site having a specific RNA sequence within the packaging region of a lentiviral vector genome molecule having a mutated MSD, which has been pre-selected as a target site for binding / annealing of a modified U1snRNA.

[0384] As used herein, the terms "packaging region of an MSD-mutated lentiviral vector genome molecule" and "packaging region of an MSD-mutated lentiviral vector genome sequence" refer to the region at the 5' end of an MSD-mutated lentiviral vector genome from the beginning of the 5' U5 domain to the end of the sequence derived from the gag gene. Thus, the packaging region of an MSD-mutated 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 sequences derived from the gag gene. It is common in the art to provide an entire 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 and can be codon-optimized; importantly, the primary attribute 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" may refer to the region at the 5' end of the lentiviral vector genome molecule from the beginning of the 5'U5 domain to the "core" packaging signal of the SL3ψ element in which the MSD has been mutated, 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.

[0385] As used herein, the term "sequence derived from the gag gene" refers to any naturally occurring sequence of the gag gene derived from nucleotide 688 from the ATG codon that may be present, e.g., may remain, in the vector genome (Kharytonchyk, S. et al., 2018, J. Mol. Biol., 430:2066-79).

[0386] As used herein, the terms "introducing a heterologous sequence within the first 11 nucleotides of U1 snRNA, including the natural splice donor annealing sequence," "introducing said heterologous sequence within the 9 nucleotides from positions 3 to 11," and "introducing a heterologous sequence within the first 11 nucleotides of the 5' end of U1 snRNA" include replacing all or part of the first 11 nucleotides or the 9 nucleotides from positions 3 to 11 of U1 snRNA with said heterologous sequence, or modifying the first 11 nucleotides or the 9 nucleotides from positions 3 to 11 of U1 snRNA so that they have the same sequence as the heterologous sequence.

[0387] As used herein, the terms "introducing a heterologous sequence into a native splice donor annealing sequence" and "introducing a heterologous sequence into a native splice donor annealing sequence at the 5' end of U1 snRNA" include replacing all or part of the native splice donor annealing sequence with said heterologous sequence, or modifying the native splice donor annealing sequence to have the same sequence as said heterologous sequence.

[0388] As used herein, the term "enhancing lentiviral vector titer" includes "increasing lentiviral vector titer," "restoring lentiviral vector titer," and "improving lentiviral vector titer."

[0389] Thus, in one embodiment, the modified U1 snRNA is modified to bind the MSD to a nucleotide sequence within the packaging region of the mutated lentiviral vector genome sequence.

[0390] 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 MSD-mutated lentiviral vector genome.

[0391] 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 MSD-mutated lentiviral vector genome.

[0392] The modified U1 snRNA may be modified at the 5' end relative to the endogenous U1 snRNA to replace the sequence encompassing the natural splice donor annealing sequence with a heterologous sequence complementary to said nucleotide sequence.

[0393] The modified U1 snRNA may be a modified U1 snRNA variant. The U1 snRNA variant modified according to the present invention may 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 may be U1_m1 or U1_m2, preferably U1A_m1 or U1A_m2.

[0394] In some embodiments, the modified U1 snRNA described herein 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, the 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 is as follows:

[0395] GCAGGGGAGATACCATGATCACGAAGGTGGTTTTCCCAGGGCGAGGCTTATCCATTGCACTCCGGATGTGCTGACCCCTGCGATTTCCCCAAATGTGGGAAACTCGACTGCATAATTTGTGGTAGTGGGGGACTGCGTTCGCGCTTTCCCCTG. (SEQ ID NO: 131) In some preferred embodiments, the first 11 nucleotides of U1 snRNA, including the natural splice donor annealing sequence, may be replaced in whole or in part with a heterologous sequence complementary to a nucleotide sequence in the packaging region of a lentiviral vector genome in which the MSD has been mutated. Suitably, nucleic acids 1 to 11 (suitably 2 to 11, 3 to 11, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) of the first 11 nucleotides of U1 snRNA are replaced with a heterologous sequence complementary to a nucleotide sequence in the packaging region of a lentiviral vector genome in which the MSD has been mutated.

[0396] In some embodiments, the native splice donor annealing sequence may be replaced in whole or in part with a heterologous sequence complementary to a nucleotide sequence in the packaging region of a lentiviral vector genome in which the MSD has been mutated. 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 with a heterologous sequence complementary to a nucleotide sequence in the packaging region of a lentiviral vector genome in which the MSD has been mutated. In a preferred embodiment, the entire native splice donor annealing sequence is replaced with a heterologous sequence complementary to a nucleotide sequence in the packaging region of a lentiviral vector genome in which the MSD has been mutated, i.e., the native splice donor annealing sequence (e.g., 5'-ACUUACCUG-3') is completely replaced with a heterologous sequence as described herein.

[0397] In some embodiments, the heterologous sequence complementary to a nucleotide sequence within the packaging region of a lentiviral vector genome having a mutated MSD comprises at least 7 nucleotides complementary to said nucleotide sequence. In some embodiments, the heterologous sequence complementary to a nucleotide sequence within the packaging region of a lentiviral vector genome having a mutated MSD comprises at least 9 nucleotides complementary to said nucleotide sequence. Preferably, the heterologous sequence for use in the present invention comprises 15 nucleotides complementary to said nucleotide sequence.

[0398] 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.

[0399] Suitably, a heterologous sequence for use in the present invention may comprise 25 nucleotides.

[0400] In some embodiments, the nucleotide sequence in the packaging region of the lentiviral vector genome with a mutated MSD is located within a sequence derived from the 5'U5 domain, the PBS element, the SL1 element, the SL2 element, the SL3ψ element, the SL4 element, and / or the gag gene. Suitably, the nucleotide sequence in the packaging region of the lentiviral vector genome with a mutated MSD 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 with a mutated MSD 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 with a mutated MSD is located within the SL1 element.

[0401] In some embodiments, the nucleotide sequence within the packaging region of the MSD-mutated lentiviral vector genome comprises at least 7 nucleotides. In some embodiments, the nucleotide sequence within the packaging region of the MSD-mutated lentiviral vector genome comprises at least 9 nucleotides. Suitably, the nucleotide sequence within the packaging region of the MSD-mutated lentiviral vector genome 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.

[0402] Preferably, the nucleotide sequence within the packaging region of the MSD-mutated lentiviral vector genome comprises 15 nucleotides.

[0403] Binding of the modified U1snRNA described herein to a nucleotide sequence within the packaging region of a lentiviral vector genome in which MSD has been mutated can enhance lentiviral vector titer during lentiviral vector production compared to lentiviral vector production in the absence of the modified U1snRNA described herein. Thus, producing a lentiviral vector in the presence of the modified U1snRNA described herein enhances lentiviral vector titer compared to lentiviral vector production in the absence of the modified U1snRNA described herein. Suitable assays for measuring lentiviral vector titer are described herein. Suitably, lentiviral vector production involves coexpression of the modified U1snRNA with vector components including gag, env, rev, and the lentiviral vector RNA genome. The lentiviral vector RNA genome can be the MSD-2KO RNA genome. In some embodiments, enhanced lentiviral vector titer occurs in the presence or absence of a functional 5'LTR polyA site. In some embodiments, the enhanced lentiviral vector titer mediated by the modified U1 snRNA of the present invention is independent of polyA site suppression in the 5'LTR of the vector genome.

[0404] In some embodiments, binding of the modified U1snRNA described herein to a nucleotide sequence within the packaging region of a lentiviral vector genome having a mutated MSD can increase lentiviral vector titer during lentiviral vector production by at least 30% compared to lentiviral vector production in the absence of the modified U1snRNA described herein. Suitably, binding of the modified U1 snRNA described herein to a nucleotide sequence within the packaging region of an MSD-mutated lentiviral vector genome may increase the titer of the MSD-mutated lentiviral vector during production by at least 35% (suitably 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%, 1,000%, 2,000%, 5,000%, or 10,000%) compared to MSD-mutated lentiviral vector production in the absence of the modified U1 snRNA described herein.

[0405] The modified U1 snRNA described herein can be designed by (a) selecting a target site (a preselected nucleotide site) within the packaging region of a lentiviral vector genome mutated in the MSD for binding of the modified U1 snRNA, and (b) introducing a heterologous sequence complementary to the preselected nucleotide site selected in step (a) within the natural splice donor annealing sequence (e.g., 5'-ACUUACCUG-3') at the 5' end of the U1 snRNA.

[0406] It is within the ability of one of ordinary skill in the art to use conventional techniques in molecular biology to introduce a complementary heterologous sequence into or in place of the natural splice donor annealing sequence (e.g., 5'-ACUUACCUG-3') at the 5' end of endogenous U1 snRNA at a target site. Generally speaking, suitable routine methods include directed mutagenesis or replacement by homologous recombination.

[0407] It is within the ability of one of ordinary skill in the art to use conventional techniques in molecular biology to modify the natural splice donor annealing sequence (e.g., 5'-ACUUACCUG-3') at the 5' end of endogenous U1 snRNA to have the same sequence as the heterologous sequence complementary to the target site. For example, suitable methods include directed or random mutagenesis followed by selection of mutations that result in the modified U1 snRNA described herein.

[0408] The modified U1 snRNA described herein 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.

[0409] In one aspect, the nucleotide sequence encoding the modified U1 snRNA can be present on a different nucleotide sequence, for example on a different plasmid.

[0410] It is within the capabilities of one of ordinary skill in the art to introduce into cells nucleotide sequences encoding the modified U1 snRNA described herein using conventional molecular and cell biology techniques.

[0411] vector Another aspect of the present invention relates to a viral vector comprising a nucleic acid sequence of the present invention.

[0412] A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. According to the present invention, for example, some vectors used in recombinant nucleic acid technology allow an entity such as a nucleic acid segment (e.g., a heterologous DNA segment, such as a heterologous cDNA segment) to be introduced into a target cell. A vector can serve the purpose of maintaining heterologous nucleic acid (DNA or RNA) in a cell, or of promoting the replication of a vector containing a DNA or RNA segment or the expression of a protein encoded by the nucleic acid segment.

[0413] The vector of the present invention may be, for example, a viral vector, which may be provided with a replication origin, a promoter for expressing the polynucleotide, and a promoter regulator. The vector may contain one or more selectable marker genes (e.g., a neomycin resistance gene) and / or a traceable marker gene (e.g., a gene encoding GFP). The vector may be used, for example, to infect and / or transduce target cells.

[0414] The vectors of the present invention can be used to replicate the NOI in compatible target cells in vitro. Thus, the present invention provides a method for producing a protein in vitro by introducing the vectors of the present invention into compatible target cells in vitro and growing the target cells under conditions that result in expression of the NOI. The protein 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.

[0415] The vector can be an expression vector.The expression vector described herein comprises a region of nucleic acid that contains a sequence that can be transcribed.Therefore, the sequence that encodes mRNA, tRNA and rRNA is included in this definition.Preferably, the expression vector comprises the polynucleotide of the present invention operably linked to a control sequence that can provide the expression of the coding sequence by target cells.

[0416] viral vectors In one embodiment of the present invention, the vector is a viral vector, which may also be referred to as a vector, vector virion, or vector particle.

[0417] In one embodiment, the viral vector is produced by a viral vector production system described herein.

[0418] In one aspect the viral vector comprises more than one NOI, at least one NOI operably linked to a tbs or part thereof as described herein.

[0419] In another embodiment, the viral vector is derived from a retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, or baculovirus.

[0420] It is anticipated that the suppression system of the present invention will be beneficial for any viral vector system, and will find particular application where the nucleotide of interest causes a deleterious effect, for example, on the viral vector producing cell or during virion assembly.

[0421] In another embodiment, the retrovirus is derived from a foamy virus.

[0422] In another embodiment, the retroviral vector is derived from a lentivirus.

[0423] In another embodiment, the lentiviral vector is derived from an HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or visna lentivirus.

[0424] Vector titer Those skilled in the art will appreciate that there are several different methods for determining the titer of a viral vector. Titer is often described as transducing units / mL (TU / mL). Titer can be increased by increasing the number of infectious particles and by increasing the specific activity of the vector preparation.

[0425] Retroviral and lentiviral vectors The retroviral vectors of the present invention can be derived from or derivable from any suitable retrovirus. Many different retroviruses have been identified. Examples include murine leukemia virus (MLV), human T-cell leukemia virus (HTLV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (MoMLV), FBR murine osteosarcoma virus (FBRMSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myelocytomatosis virus-29 (MC29), and avian erythroblastosis virus (AEV). A comprehensive list of retroviruses can be found in Coffin et al. (1997) "Retroviruses," Cold Spring Harbor Laboratory Press, Eds: JM Coffin, SM Hughes, H.E. Varmus, pp. 758-763.

[0426] Retroviruses can be broadly divided into two categories: "simple" and "complex." Retroviruses can be further divided into seven groups. Five of these groups correspond to retroviruses with oncogenic potential. The remaining two groups are lentiviruses and spumaviruses. A review of these retroviruses is presented in Coffin et al. (1997) ibid.

[0427] The basic genome structures of retroviruses and lentiviruses share many common features, such as a packaging signal that allows the genome to be packaged, a primer binding site, an integration site that allows integration into the target cell genome, and 5'LTR and 3'LTR located between or within the gag / pol and env genes that encode packaging components - polypeptides required for viral particle assembly. Lentiviruses have additional features, such as the rev gene and RRE sequence in HIV, that allow efficient export of integrated proviral RNA transcripts from the nucleus to the cytoplasm of infected target cells.

[0428] In the provirus, these genes are flanked at both ends by regions called long terminal repeats (LTRs). The LTRs are responsible for the integration and transcription of the provirus. The LTRs also function as enhancer-promoter sequences, which can control the expression of viral genes.

[0429] The LTR itself is an identical sequence that 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 the three elements can vary considerably between different retroviruses.

[0430] In a typical retroviral vector of the present invention, 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.

[0431] Portions of the viral genome can also be replaced with libraries encoding the candidate nucleic acid binding sequences described herein operably linked to regulatory control regions and reporter genes within the vector genome to generate vectors containing the candidate nucleic acid binding sequences described herein that are capable of transducing target non-dividing cells and / or integrating their genome into the host genome.

[0432] Lentiviruses are part of the larger group known as 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 prototypical "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), and bovine immunodeficiency virus (BIV).

[0433] 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 J11(8):3053-3058 and Lewis and Emerman (1994) J Virol 68(1):510-516). In contrast, other retroviruses, such as MLV, are unable to infect non-dividing or slowly dividing cells, such as those that make up muscle, brain, lung, and liver tissue.

[0434] A lentiviral vector, as used herein, is a vector that contains at least one component moiety derivable from a lentivirus, preferably the component moiety being involved in the biological mechanisms by which the vector infects cells, expresses genes, or replicates.

[0435] Lentiviral vectors can be derived from either primate lentiviruses (eg, HIV-1) or non-primate lentiviruses.

[0436] Examples of non-primate lentiviruses may be any member of the Lentiviridae family that does not naturally infect primates, and may include feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), caprine arthritis-encephalitis virus (CAEV), Maedi-Visna virus (MVV), or equine infectious anemia virus (EIAV).

[0437] Generally speaking, a typical retroviral vector production system involves the separation of the viral genome from the essential viral packaging functions. These components are typically provided to the producer cell on a separate DNA expression cassette (alternatively known as a plasmid, expression plasmid, DNA construct, or expression construct).

[0438] The vector genome contains an NOI. A vector genome typically requires an internal expression cassette containing a packaging signal (ψ), a central polypurine tract (cppt), a rev-responsive element (RRE), and an NOI. A post-transcriptional element (PRE), typically a central polypurine tract (cppt), a 3'-ppu, and a self-inactivating (SIN) long-term repeat (LTR), may be required. The R-U5 region is required for correct polyadenylation of both the vector genome RNA and the NOI mRNA, as well as for the process of reverse transcription. A vector genome may also contain an open reading frame, as described in International Publication No. WO 2003 / 064665.

[0439] In one aspect, the nucleotide sequence can be suitable for use in lentiviral vectors in Tat-independent systems for vector production.As described herein, third-generation lentiviral vectors are Tat-independent, and the nucleotide sequence according to the present invention can be used in the context of third-generation lentiviral vectors.In one aspect of the present invention, Tat is not provided in the lentiviral vector production system, for example, Tat is not provided in trans.In one aspect, the cells or vectors or vector production systems described herein do not contain Tat protein.

[0440] Packaging functions include the gag / pol and env genes, which are required for production of vector particles by producer cells. Providing these functions in trans into the genome facilitates the production of replication-deficient viruses.

[0441] Production systems for gammaretroviral vectors are typically three-component systems requiring the genome, gag / pol, and env expression constructs. Production systems for HIV-1-based lentiviral vectors additionally require that the accessory gene rev be provided in trans, and that the vector genome contain a rev response element (RRE). EIAV-based lentiviral vectors do not require rev if an open reading frame (ORF) is present (see WO 2003 / 064665).

[0442] Typically, both the "external" promoter (driving the vector genome cassette) and the "internal" promoter (driving the NOI cassette) encoded within the vector genome cassette are strong eukaryotic or viral promoters, as are the promoters driving other vector system components. Examples of such promoters include CMV, EF1α, PGK, CAG, TK, SV40, and ubiquitin promoters. Strong "synthetic" promoters, such as those generated by DNA libraries (e.g., the JeT promoter), can also be used to drive transcription. Alternatively, to drive transcription, 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, neuronal-specific enolase (NSE) promoter, astrocyte-specific glial fibrillary acidic protein (GFAP) promoter, human α1-antitrypsin (hAAT) promoter, phosphoenolpyruvate carboxykinase (PEPCK), liver fatty acid-binding protein promoter, Fl Tissue-specific promoters such as the t-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 may be used.

[0443] Production of retroviral vectors involves either transient transfection of producer cells with these DNA components or the use of stable producer cell lines (PCLs) in which the components have been integrated into the producer cell genome (e.g., Stewart, HJ, MALeroux-Carlucci, CJ Schion, KAMiotrophanous, and P Aadcliffe (2009) Gene Ther. 16(6):805-814 Epub 2009 Mar 2005). Another approach is to use stable packaging cells (in which the packaging components have been stably integrated) and then transiently transfect them with a vector genome plasmid, if desired. To generate the viral vectors of the present invention, the producer cells must be capable of expressing TRAP. Thus, in one embodiment of the present invention, the producer cells will stably express the TRAP construct. In another embodiment of the present invention, the producer cells will transiently express the TRAP construct. In another embodiment of the present invention, the producer cells will stably express the TRAP construct and also transiently express the TRAP construct.

[0444] It should be noted that although the TRIP system has been primarily described for producing retroviral vectors, similar strategies can be applied to other viral vectors.

[0445] 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 coordinates 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, an EIAV protein, Ttm, has been identified that is encoded by the first exon of tat, which is spliced ​​into the env coding sequence at the beginning of the transmembrane protein. In an alternative embodiment of the invention, the viral vector is derived from HIV: HIV differs from EIAV in that it does not encode S2, but, unlike EIAV, encodes vif, vpr, vpu, and nef.

[0446] The term "recombinant retroviral or lentiviral vector" (RRV) refers to a vector that has sufficient retroviral genetic information to allow packaging of the RNA genome into viral particles that, in the presence of packaging components, can infect target cells. Infection of the target cell can involve reverse transcription and integration into the target cell genome. RRVs carry non-viral coding sequences to be delivered to the target cell by the vector. RRVs are incapable of independent replication to produce infectious retroviral particles within the target cell. Typically, RRVs lack functional gag / pol and / or env genes and / or other genes essential for replication.

[0447] Preferably, the RRV vector of the invention has a minimal viral genome.

[0448] 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 functions required for infecting, transducing, and delivering a nucleotide sequence of interest to target cells. Further details of this strategy can be found in International Publication Nos. 1998 / 17815 and 99 / 32646. The minimal EIAV vector lacks the tat, rev, and S2 genes, none of which are provided in trans in the production system. The minimal HIV vector lacks vif, vpr, vpu, tat, and nef.

[0449] However, the expression plasmid used to produce the vector genome in producer cells will contain transcriptional regulatory control sequences operably linked to the retroviral genome to direct transcription of the genome in the producer / packaging cells. These regulatory sequences may be natural sequences associated with the retroviral sequence to be transcribed, i.e., the 5' U3 region, or, as described below, may be another viral promoter, such as a heterologous promoter, such as the CMV promoter. 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 RRE (and the dependency on rev provided in trans) can be reduced or eliminated by codon optimization. Further details of this strategy can be found in WO 2001 / 79518. Other 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 is known as a constitutive transport element (CTE), and it contains an RRE-type sequence in the genome that is thought to interact with factors in infected cells. The cellular factor can be considered a rev analog. Thus, CTE can be used as a substitute for the rev / RRE system. Any other functional equivalent that is known or becomes available may be suitable for the present invention. For example, it is also known that the Rex protein of HTLV-I can functionally replace the Rev protein of HIV-1. Rev and RRE may be absent or non-functional in the vector for use in the method of the present invention, and alternative rev and RRE, or functionally equivalent systems, may exist.

[0450] SIN vector Vectors for use in the methods of the present invention are preferably used in a self-inactivating (SIN) configuration in which viral enhancer and promoter sequences are deleted. SIN vectors can be engineered to transduce non-dividing target cells in vivo, ex vivo, or in vitro with efficacy similar to that of wild-type vectors. Transcriptional inactivation of the long terminal repeat (LTR) 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 gene expression from internal promoters by eliminating any cis-acting effects of the LTR.

[0451] 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 a series of vector reverse transcription and integration, these changes are replicated into both the 5' and 3' LTRs, producing a transcriptionally inactive "provirus." However, any promoter(s) within the LTR in such vectors remain transcriptionally active. This strategy has been used to eliminate the influence of enhancers and promoters in the viral LTR on transcription from internally placed genes. Such effects include increased transcription or repression of transcription. This strategy can also be used to eliminate downstream transcription from the 3' LTR into genomic DNA. This is of particular concern 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 U.S. Patent No. 6,924,123 and U.S. Patent No. 7,056,699.

[0452] Non-replicating lentiviral vectors In the genome of a replication-deficient lentiviral vector, the gag / pol and / or env sequences may be mutated, absent, and / or non-functional.

[0453] In the typical lentiviral vector of the present invention, at least a portion of the coding region of one or more proteins essential for viral replication can be removed from the vector.This makes the viral vector replication-defective.A portion of the viral genome can also be replaced with a nucleotide of interest (NOI) to create a vector containing a NOI that can transduce non-dividing target cells and / or integrate its genome into the target cell genome.

[0454] In one embodiment, the lentiviral vector is a non-integrating vector such as those described in WO 2006 / 010834 and WO 2007 / 071994.

[0455] In a further embodiment, the vector has the ability to deliver sequences that lack or lack viral RNA. In a further embodiment, to ensure the packaging of the RNA to be delivered, 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. Both of these vectors are described in WO2007 / 072056.

[0456] Adenovirus vectors In another embodiment of the present invention, vector can be adenovirus vector.Adenovirus is a double-stranded linear DNA virus that does not replicate through RNA intermediate.There are more than 50 different human serotypes of adenovirus, which are divided into 6 subgroups based on their gene sequence.

[0457] Adenoviruses are non-enveloped, double-stranded DNA viruses capable of in vivo, ex vivo, and in vitro transduction of a wide range of cell types of human and non-human origin, including post-mitotically terminally differentiated cells such as airway epithelial cells, hepatocytes, muscle cells, cardiac myocytes, synoviocytes, primary mammary epithelial cells, and neurons.

[0458] Adenoviral vectors can also transduce non-dividing cells. This is crucial for diseases such as cystic fibrosis, in which affected cells within the lung epithelium have a slow turnover rate. Indeed, several trials are underway utilizing adenovirus-mediated delivery of the cystic fibrosis transporter (CFTR) into the lungs of adult cystic fibrosis patients.

[0459] Adenoviruses have been used as vectors for gene therapy and expression of heterologous genes. Their large (36 kb) genome can accommodate up to 8 kb of foreign DNA insert and replicates efficiently in complementing cell lines, resulting in up to 10 12 Very high titers of transducing units / ml can be produced, making adenoviruses one of the best systems for studying gene expression in primary non-replicating cells.

[0460] Expression of viral or foreign genes from the adenoviral genome does not require a replicating cell. Adenoviral vectors enter cells through receptor-mediated endocytosis. Once inside the cell, adenoviral vectors rarely integrate into the host chromosome. Instead, adenoviral vectors function episomally (independently of the host genome) as linear genomes within the host nucleus.

[0461] Adeno-associated virus vector Adeno-associated virus (AAV) has high integration frequency and can infect non-dividing cells, so it is an attractive vector system for use in the present invention.Therefore, adeno-associated virus (AAV) is useful for delivering genes into mammalian cells.AAV has a wide host range for infection.The details of the generation and use of rAAV vector are described in United States Patent No. 5,139,941 and United States Patent No. 4,797,368, each of which is incorporated herein by reference.

[0462] Recombinant AAV vectors have been used successfully for in vitro, ex vivo, and in vivo transduction of marker genes and genes involved in human diseases.

[0463] Specific AAV vectors have been developed that can efficiently integrate large payloads (up to 8-9 kb). One such vector has an AAV5 capsid and AAV2 ITRs (Allocca M, et al. J. Clin Invest (2008) 118:1955-1964).

[0464] Herpes simplex virus vector Herpes simplex virus (HSV) is an enveloped, double-stranded DNA virus that naturally infects neural cells. HSV can accommodate large segments of foreign DNA, making it attractive as a vector system and has been used as a vector for gene delivery to neural cells (Manservigiet et al. Open Virol J. (2010) 4:123-156).

[0465] The use of HSV in therapeutic procedures requires that the strain be attenuated so that it cannot establish a lytic cycle.In particular, when HSV vectors are used for human gene therapy, polynucleotides should preferably be inserted into essential genes.This is because when viral vectors encounter wild-type viruses, recombination can lead to the introduction of heterologous genes into wild-type viruses.However, as long as polynucleotides are inserted into essential genes, this recombination will also delete essential genes in recipient viruses, preventing the "escape" of heterologous genes into the wild-type virus population that has replication capacity.

[0466] Vaccinia virus vector The vectors of the invention can be vaccinia virus vectors such as MVA or NYVAC. Alternatives to vaccinia vectors include avian pox vectors, such as fowlpox or canarypox, known as ALVAC, and strains derived therefrom that are capable of infecting and expressing recombinant proteins in human cells but are unable to replicate.

[0467] Baculovirus vectors The vector of the present invention can also be a baculovirus vector.The modification of baculovirus to allow the expression of the encoded NOI in mammalian cells is well known in the art.This can be achieved, for example, by using a mammalian promoter upstream of the NOI.

[0468] Vectors encoding multiple NOIs In one aspect the vector comprises more than one NOI, wherein one or more NOIs are operably linked to a tbs or part thereof as described herein.

[0469] Internal ribosome entry site (IRES) As mentioned above, a vector of the invention may contain more than one NOI, and to express these NOIs there may be two or more transcription units within the vector genome, one for each NOI. However, it is clear from the literature that retroviral vectors achieve the highest titers and most potent gene expression characteristics if they are kept genetically simple (WO 96 / 37623; Bowtell et al., 1988 J. Virol. 62, 2464; Correll et al., 1994 Blood 84, 1812; Emerman and Temin 1984 Cell 39, 459; Ghattas et al., 1991 Mol. Cell. Biol. 11, 5848; Hantzopoulos et al., 1989 PNAS 86, 3519; Hatzoglou et al., 1991 J. Biol. Chem 266, 8416; Hatzoglou et al., 1988 J. Biol. Chem 263, 17798; Li et al., 1992 Hum. Gen. Ther. 3, 381; McClachlin et al., 1993 Virol. 195, 1; Overell et al., 1988 Mol. Cell Biol. 8, 1803; Scharfman et al., 1991 PNAS 88, 4626; Vile et al., 1994 Gene Ther 1, 307; Xu et al., 1989 Virol. 171, 331; Yee et al., 1987 PNAS 84, 5197), and therefore it is preferable to use an internal ribosome entry site (IRES) to initiate translation of the second (and subsequent) coding sequence in a polycistronic message (Adam et al 1991 J. Virol. 65, 4985).

[0470] Insertion of an IRES element into a retroviral vector is compatible with the retroviral replication cycle and 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.

[0471] A review on IRES is presented by 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 Drosophila antennapedia gene (exons d and e) [Oh, et al., Genes & Development, 6:1643-1653 (1992)], as well as in poliovirus (PV) [Pelletier and Sonenberg, Nature 334:320-325 (1988); see also Mountford and Smith, TIG 11, 179-184 (1985)].

[0472] IRES elements from PV, EMCV and swine vesicular disease virus have been used previously in retroviral vectors (Coffin et al., supra).

[0473] The term "IRES" includes any sequence or combination of sequences that functions as an IRES or improves the function of an IRES.

[0474] The IRES(s) may be of viral origin (such as the EMCV IRES (SEQ ID NO: 59), PV IRES or FMDV 2A-like sequence) or cellular origin (such as the FGF 2 IRES, NRF IRES, Notch 2 IRES or EIF4 IRES).

[0475] The IRES should be located between or before the polynucleotides in the vector genome so that the IRES is able to initiate translation of each polynucleotide.

[0476] promoter The expression of the NOI can be controlled using control sequences including promoters / enhancers and other expression regulation signals. Prokaryotic promoters and promoters functional 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.

[0477] 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 from heterologous mammalian promoters such as the actin promoter, EF1α, CAG, TK, SV40, ubiquitin, PGK or ribosomal protein promoters. Alternatively, to drive transcription, 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, neuronal-specific enolase (NSE) promoter, astrocyte-specific glial fibrillary acidic protein (GFAP) promoter, human α1-antitrypsin (hAAT) promoter, phosphoenolpyruvate carboxykinase (PEPCK), liver fatty acid-binding protein promoter, Fl Tissue-specific promoters such as the t-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 may be used.

[0478] Gene transcription can be further increased by inserting an enhancer sequence into the vector. Enhancers are relatively orientation- and position-independent. However, enhancers from eukaryotic viruses, such as the SV40 enhancer on the late side of the replication origin (bp 100-270) and the CMV early promoter enhancer, can be used. The enhancer can be spliced ​​into the vector at a 5' or 3' position relative to the promoter, but is preferably located at a site 5' from the promoter.

[0479] The promoter may further include features to ensure or increase expression in appropriate target cells. For example, features may be conserved regions, such as a Pribnow box or a TATA box. The promoter may contain other sequences to affect (e.g., maintain, enhance, or decrease) the level of expression of the nucleotide sequence. Suitable other sequences include the Sh1 intron or the ADH intron. Other sequences include inducible elements, such as temperature, chemical, light, or stress-inducible elements. Suitable elements for enhancing transcription or translation may also be present.

[0480] The TRAP-tbs interaction may be useful in forming the basis for a transgene protein suppression system for the production of retroviral vectors when a constitutive and / or strong promoter driving the transgene is desirable, including a tissue-specific promoter, particularly when expression of the transgene protein in producer cells results in reduced vector titer and / or elicits an immune response in vivo due to viral vector delivery of the transgene-derived protein.

[0481] Regulators of NOIs A complicating factor in the generation of retroviral packaging / producer cell lines and retroviral vector production is the inability to produce vectors because constitutive expression of certain retroviral vector components and NOIs is cytotoxic, resulting in the death of cells expressing these components. Therefore, expression of these components (e.g., envelope proteins such as gag-pol and 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, modular constructs or nucleotide sequences encoding the vector components and / or cells described herein can contain cytotoxic and / or non-cytotoxic vector components with at least one regulatory element. As used herein, the term "regulatory element" refers to any element that can affect, increase, or decrease the expression of an associated gene or protein. Regulatory elements include gene switch systems, transcriptional regulatory elements, and translational repression elements.

[0482] Many prokaryotic regulatory element systems have been developed to generate gene switches in mammalian cells. Many retroviral packaging and producer cell lines are controlled using gene switch systems (e.g., tetracycline and cumate-inducible switch systems) to switch on the expression of one or more retroviral vector components during vector production. Gene switch systems include gene switch systems based on the TetR protein family of transcriptional regulators (e.g., T-Rex, Tet-On, and Tet-Off), gene switch systems based on the cumate-inducible switch family of transcriptional regulators (e.g., CymR protein), and gene switch systems involving RNA-binding proteins (e.g., TRAP).

[0483] 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), and then TetR alone can act 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 the NOI can be controlled by a CMV promoter into which two copies of the TetO2 sequence have been inserted in tandem. In the absence of an inducer (tetracycline or its analog doxycycline [dox]), the TetR homodimer binds to the TetO2 sequence and physically blocks transcription from the upstream CMV promoter. In the presence of an inducer, the inducer binds to the TetR homodimer, causing an allosteric change such that the TetR homodimer can no longer bind to the TetO2 sequence, resulting in gene expression. Because codon optimization has been found to improve translation efficiency, resulting in tighter control of gene expression controlled by TetO2, the TetR gene can be codon-optimized.

[0484] The TRiP system, described in International Publication No. 2015 / 092440, provides another method for suppressing expression of NOIs in producer cells during vector production. When a constitutive and / or strong promoter, including a tissue-specific promoter, driving a transgene is desirable, particularly when expression of the transgene protein in the producer cell results in reduced vector titer and / or induces an immune response in vivo due to viral vector delivery of the transgene-derived protein, TRAP-binding sequence (e.g., TRAP-tbs) interaction forms the basis for a transgene protein suppression system for the production of retroviral vectors (Maunder et al., Nat Commun. (2017) Mar 27;8).

[0485] Briefly, the TRAP-tbs interaction forms a translation block, suppressing the translation of the transgene protein (Maunder et al., Nat Commun. (2017) Mar 27;8). The translation block is only effective in producer cells and therefore does not interfere with DNA- or RNA-based vector systems. The TRiP system can suppress translation when the transgene protein is expressed from a constitutive and / or strong promoter, including tissue-specific promoters from mono- or bicistronic mRNAs. It has been demonstrated that unregulated expression of the transgene protein can reduce vector titer and affect the quality of the vector product. Suppression of transgene proteins for both transient and stable PaCL / PCL vector production systems is beneficial to prevent producer cells from reducing vector titers when toxicity or molecular burden issues can result in cellular stress; when transgene proteins induce immune responses in vivo due to viral vector delivery of transgene-derived proteins; when the use of gene editing transgenes can result in on-target / off-target effects; or when transgene proteins can affect the clearance of vectors and / or envelope glycoproteins.

[0486] Packaging Sequence As utilized within the context of the present invention, the term "packaging signal," interchangeably referred to as "packaging sequence" or "psi," 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 has been mapped to a locus extending from upstream of the major splice donor site (SD) to at least the gag initiation codon. In EIAV, the packaging signal comprises an R region in the 5' coding region of Gag.

[0487] As used herein, the term "extended packaging signal" or "extended packaging sequence" refers to the use of sequences surrounding the psi sequence with additional extensions in the gag gene. The inclusion of these additional packaging sequences can increase the efficiency of insertion of vector RNA into viral particles.

[0488] The feline immunodeficiency virus (FIV) RNA encapsidation determinants have been shown to be separate and non-contiguous, comprising one region (R-U5) at the 5' end of the genomic mRNA and another region mapped within the proximal 311 nt of gag (Kaye et al., J Virol. Oct;69(10):6588-92 (1995)).

[0489] pseudotyping In one preferred aspect, the viral vector of the present invention is pseudotyped. In this regard, pseudotyping can provide one or more advantages. For example, the env gene product of HIV-based vectors restricts these vectors to infect only cells that express a protein called CD4. However, if the env gene in these vectors is replaced with an env sequence from another enveloped virus, these vectors 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).

[0490] 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).

[0491] Vectors can be pseudotyped with any molecule of choice.

[0492] 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.

[0493] The ability to pseudotype MoMLV-based retroviral vectors in the absence of any retroviral envelope proteins was first demonstrated by Emi et al. (1991) Journal of Virology 65:1202-1207). International Publication No. 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. More recently, Abe et al. (1998) J Virol 72(8)6356-6361 teaches that the addition of VSV-G can make non-infectious retroviral particles infectious.

[0494] 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 its native 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) ibid.). VSV-G pseudotyped vectors have also been shown to be more efficient than conventional amphotropic envelopes against a variety of 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 because its cytoplasmic tail can interact with the retroviral core.

[0495] Pseudotyping a nonretroviral envelope, such as the VSV-G protein, offers the advantage of allowing vector particles to be concentrated to high titers without loss of infectivity (Akkina et al. (1996) J. Virol. 70:2581-5). Presumably consisting of two noncovalently linked subunits, retroviral envelope proteins apparently cannot withstand the shear forces during ultracentrifugation. Intersubunit interactions can be disrupted by centrifugation. In comparison, the VSV glycoprotein consists of a single unit. Therefore, pseudotyping VSV-G protein offers potential advantages.

[0496] WO 2000 / 52188 describes the production of pseudotyped retroviral vectors from stable producer cell lines carrying the vesicular stomatitis virus-G protein (VSV-G) as a membrane-bound viral envelope protein and provides the gene sequence for the VSV-G protein.

[0497] Ross River virus The Ross River Virus envelope has been used to pseudotype non-primate lentiviral vectors (FIV) and primarily transduced 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 vectors pseudotyped with VSV-G and to have caused less cytotoxicity, as measured by serum levels of liver enzymes indicative of hepatotoxicity.

[0498] Baculovirus GP64 The baculovirus GP64 protein has been shown to be an alternative to VSV-G for viral vectors used in 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 with vectors pseudotyped with VSV-G, vectors pseudotyped with GP64 have similar broad tropism and similar natural titers. Because GP64 expression does not kill cells, 293T-based cell lines can be generated that constitutively express GP64.

[0499] 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.

[0500] Viral vector production systems and cells Another aspect of the present invention relates to a viral vector production system comprising a set of nucleic acid sequences encoding components required for the production of a viral vector, wherein the vector genome sequence comprises a nucleic acid sequence of the present invention.

[0501] A "viral vector production system" or "vector production system" or "production system" should be understood as a system that contains the components necessary for viral vector production.

[0502] Therefore, the vector production system comprises a set of nucleic acid sequences encoding the components necessary to produce viral vector particles. One such nucleic acid sequence may comprise a gene encoding TRAP. In a preferred embodiment, the RNA-binding protein is bacterial TRAP.

[0503] In one embodiment of the present invention, the viral vector is a retroviral vector, and the viral vector production system further comprises nucleic acid sequences encoding Gag and Gag / Pol proteins and Env protein or functional substitutes thereof, and a vector genome sequence comprising a nucleic acid sequence of the present invention. The production system may also comprise a nucleic acid sequence encoding a Rev protein and / or a nucleic acid sequence encoding TRAP.

[0504] In another embodiment of the viral vector production system of the present invention, the viral vector is derived from a retrovirus, adenovirus, or adeno-associated virus.

[0505] In another embodiment, the viral vector is derived from a lentivirus. In another embodiment, the viral vector is derived from an HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or visna lentivirus.

[0506] Another aspect of the present invention relates to a method for increasing viral vector titer in a eukaryotic vector-producing cell, comprising introducing into the eukaryotic vector-producing cell a viral vector production system of the present invention and a nucleic acid sequence encoding TRAP, wherein TRAP binds to the TRAP binding site or a portion thereof and inhibits translation of the NOI, thereby increasing the viral vector titer compared to a viral vector that does not have a TRAP binding site.

[0507] Another aspect of the present invention relates to DNA constructs for use in the viral vector production system of the present invention. Such DNA constructs (e.g., plasmids) may comprise vector genome constructs comprising the nucleic acid sequences of the present invention.

[0508] A further aspect of the present invention relates to a DNA construct for use in the viral vector production system of the present invention, which comprises a nucleic acid sequence encoding TRAP.

[0509] Another aspect of the present invention relates to a set of DNA constructs for use in the viral vector production system of the present invention, comprising a DNA construct of the present invention and DNA constructs encoding Gag and Gag / Pol proteins and Env proteins or functional substitutes thereof.

[0510] In one embodiment of the present invention, the set of DNA constructs further comprises a DNA construct encoding TRAP.

[0511] In one embodiment of the present invention, the set of DNA constructs further comprises a DNA construct encoding a Rev protein or a functional substitute thereof.

[0512] In one embodiment, the viral vector production system includes a modular nucleic acid construct (modular construct). The modular construct is a DNA expression construct containing two or more nucleic acids used in the production of lentiviral vectors. The modular construct may be a DNA plasmid containing two or more nucleic acids used in the production of lentiviral vectors. The plasmid may be a bacterial plasmid. The nucleic acid may encode, for example, gag-pol, rev, env, or a vector genome. Additionally, modular constructs designed for packaging and producer cell line generation may further require encoding transcriptional regulatory proteins (e.g., TetR, CymR) and / or translational repressor proteins (e.g., TRAP) and selectable markers (e.g., Zeocin™, hygromycin, blasticidin, puromycin, neomycin resistance genes). Suitable modular constructs for use in the present invention are described in EP 3502260, the entire contents of which are incorporated herein by reference.

[0513] Because modular constructs for use in accordance with the present invention contain nucleic acid sequences encoding two or more retroviral components on a single construct, the safety profile of these modular constructs has been examined, and additional safety features have been engineered directly into the construct. These features include the use of insulators for multiple open reading frames of the retroviral vector components and / or specific orientation and placement of retroviral genes in the modular construct. The use of these features is believed to prevent direct read-through to generate replication-competent viral particles.

[0514] Nucleic acid sequences encoding viral vector components may be in reverse and / or alternate transcriptional orientations within the modular construct. Therefore, the nucleic acid sequences encoding viral vector components are not presented in the same 5' to 3' orientation, and as a result, the viral vector components cannot be produced from the same mRNA molecule. Reverse orientation may mean that at least two coding sequences for different vector components are presented in "head-to-head" and "tail-to-tail" transcriptional orientations. This can be achieved by providing a coding sequence for one vector component, e.g., env, on one strand of the modular construct and a coding sequence for another vector component, e.g., rev, on the opposite strand. Preferably, when coding sequences for more than two vector components are present in a modular construct, at least two of the coding sequences are present in reverse transcriptional orientations. Thus, when coding sequences for more than two vector components are present in a modular construct, each component may be oriented so that it is in the opposite 5' to 3' orientation relative to all of the adjacent coding sequence(s) for the other vector component(s) it is adjacent to; i.e., alternate 5' to 3' (or transcriptional) orientations may be used for each coding sequence.

[0515] Modular constructs for use in accordance with the present invention may contain nucleic acid sequences encoding two or more of the following vector components: gag-pol, rev, env, vector genome. Modular constructs may contain nucleic acid sequences encoding any combination of vector components. In one embodiment, a modular construct comprises: i) the RNA genome and rev of the retroviral vector or its functional substitute; 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 its functional substitute, and gag-pol; viii) the RNA genome of the retroviral vector, rev or its functional substitute and env; ix) the RNA genome, gag-pol and env of a retroviral vector; or x) gag-pol, rev or its functional substitute, and env; may comprise a nucleic acid sequence encoding The nucleic acid sequences may be in reverse and / or alternate orientation.

[0516] In one embodiment, the cell for producing a retroviral vector can contain nucleic acid sequences encoding any one of the combinations of i) to x) above, where the nucleic acid sequences are located at the same locus and are in opposite and / or alternate orientations. 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.

[0517] Another aspect of the present invention relates to a viral vector producing cell comprising part or all of the nucleic acid sequence, viral vector producing system or DNA construct of the present invention.

[0518] A "viral vector producing cell" should be understood as a cell capable of producing a viral vector or viral vector particle. A viral vector producing cell may be a "producer cell" or a "packaging cell." One or more DNA constructs of a viral vector system may be stably integrated into a viral vector producing cell or may be episomally maintained. Alternatively, all DNA components of the viral vector system may be transiently transfected into a viral vector producing cell. In yet another alternative, a producer cell stably expressing some of the components may be transiently transfected with the remaining components.

[0519] The DNA expression cassette encoding TRAP can be stably integrated into the viral vector-producing cells or maintained episomally, or it can be transiently transfected into the viral vector-producing cells.

[0520] Thus, in one embodiment of the invention, the production cells will stably express the TRAP construct. In another embodiment of the invention, the production cells will transiently express the TRAP construct.

[0521] Since the level of suppression required may vary depending on the NOI, the level of TRAP required in the production cells may also depend on the NOI. Thus, in some situations, a combination of stable and transient TRAP expression may be desirable. Stable expression may provide a continuous level of TRAP expression in the production cells, whereas transient expression may provide an increased level of TRAP expression for a shorter period of time. For example, suppression of more problematic / toxic transgenes may be beneficial from both existing TRAP (e.g., provided by stable expression) and high levels of TRAP during vector production.

[0522] Thus, in another embodiment of the invention, the production cells stably express the TRAP construct and also transiently express the TRAP construct, where transient expression can provide higher levels of TRAP expression for shorter periods of time than is provided by stable expression.

[0523] By "stable expression" it is understood that expression of TRAP from a construct that confers stable expression does not change substantially over an extended period of time.

[0524] By "transient expression," it is understood that expression of TRAP from a construct that confers transient expression is not stable over long periods of time. Preferably, the polynucleotide encoding the TRAP that confers transient expression is not integrated into the producer cell genome and is not maintained episomally in the producer cell.

[0525] As used herein, the term "packaging cell" refers to a cell that contains the elements necessary for the production of infectious vector particles but lacks a vector genome. Typically, such packaging cells contain one or more expression cassettes capable of expressing viral structural proteins (such as gag, gag / pol, and env).

[0526] The producer / packaging cells can be of any suitable cell type. Producer cells are generally mammalian cells, but can be, for example, insect cells.

[0527] As used herein, the terms "producer / producing cell" or "vector-producing / vector-producing cell" refer to a cell that contains all the elements necessary for the production of retroviral vector particles and expression of TRAP.

[0528] Producer cells can be either stable producer cell lines or transiently derived producer cell lines.

[0529] In one embodiment of the invention, the envelope and nucleocapsid, TRAP, and, if present, rev nucleotide sequences are all stably integrated into the producer and / or packaging cells. However, any one or more of these sequences can also be present in episomal form, and gene expression can occur from the episome or can be transiently transfected into the producer cells.

[0530] The vector-producing cells may be in vitro cultured cells, 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, the vector-producing cells are derived from human cell lines.

[0531] In one embodiment, the vector of the present invention uses as its production system four transcription units expressing a vector genome comprising a nucleic acid sequence of the present invention operably linked to an NOI, gag-pol components, an envelope, and a TRAP. The envelope expression cassette may contain one of a number of heterologous envelopes, such as VSV-G. A rev component may also be included.

[0532] Viral vector production process Another aspect of the present invention relates to a method for producing a viral vector, comprising introducing part or all of the nucleic acid sequence, viral vector production system or DNA construct of the present invention into a viral vector producer cell and culturing the producer cell under conditions suitable for production of the viral vector.

[0533] A suitable "producer cell" is a cell that can produce a viral vector or viral vector particle when cultured under appropriate conditions. Suitable "producer cells" are generally mammalian or human cells, such as HEK293T, HEK293, CAP, CAP-T, or CHO cells, but can also be insect cells, such as SF9 cells.

[0534] Producer cells may also be avian cells, such as EB66® (Sigma) cells. Avian cells may be particularly useful for the production of human and veterinary virus-based vaccines, such as influenza and Newcastle disease virus vaccines.

[0535] Methods for introducing nucleic acids into production cells are well known in the art and have been previously described.

[0536] In one embodiment, the production cell comprises a TRAP.

[0537] Another aspect of the present invention relates to a viral vector produced by the viral vector production system of the present invention, using the viral vector-producing cell of the present invention, or by the method of the present invention.

[0538] In one embodiment, viral vector particles comprise the nucleic acid sequence of the present invention.Viral vector particles can be derived from retrovirus, adenovirus or adeno-associated virus.Retroviral vector particles can be derived from lentivirus.Lentiviral vector particles can be derived from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or visna lentivirus.

[0539] Methods for producing lentiviral vectors, and in particular the processing of lentiviral vectors, are described in WO 2009 / 153563.

[0540] Another aspect of the invention pertains to cells transduced with the viral vectors of the invention.

[0541] A "cell transduced by a viral vector particle" should be understood as a cell, in particular a target cell, into which the nucleic acid carried by the viral vector particle has been introduced.

[0542] use Another aspect of the present invention relates to a viral vector of the present invention or a cell or tissue transduced with a viral vector of the present invention for use in medicine.

[0543] Another aspect of the present invention relates to the use of the viral vector of the present invention or cells or tissues transduced with the viral vector of the present invention in medicine.

[0544] Another aspect of the present invention relates to the use of a viral vector of the present invention, a producer cell of the present invention, or a cell or tissue transduced with a viral vector of the present invention for the preparation of a medicament for delivering a nucleotide of interest to a target site requiring the nucleotide of interest.

[0545] Such uses of the viral vectors or transduced cells of the invention can be for therapeutic or diagnostic purposes, as described herein.

[0546] Therapeutic Vectors Retroviral Therapeutic Vectors In one embodiment, the retroviral vector of the present invention can be used to introduce three genes encoding three enzymes in the dopamine synthesis pathway to treat Parkinson's disease. The retroviral vector is a non-replicating, self-inactivating, minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or alternative viral envelope proteins. The genes carried by the retroviral vector can include a truncated human tyrosine hydroxylase (TH*) gene (lacking the N-terminal 160 amino acids involved in the feedback regulation of TH), human aromatic L-amino acid decarboxylase (AADC), and human GTP-cyclohydrolase 1 (CH1) genes. The three enzymes can be encoded by the retroviral vector in three separate open reading frames. Alternatively, the retroviral vector can encode a fusion of the TH and CH1 enzymes in one open reading frame with the AADC enzyme in a second open reading frame. Expression of the gene can be driven by a CMV promoter, and the expression cassette can contain one or more IRES elements. Retroviral vectors can be administered by direct injection into the striatum of the brain.

[0547] In another embodiment, the retroviral vectors of the present invention can be used as gene therapy products designed to introduce a corrective MYO7A gene into photoreceptors and their supporting retinal pigment epithelial (RPE) cells, thereby attenuating or reversing the vision loss associated with Usher 1B syndrome. The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or alternative viral envelope proteins. The gene carried by the retroviral vector is MYO7A cDNA (a large gene over 100 mb in length) encoding the MYO7A protein. Expression of the large MYO7A gene can be driven by a CMV promoter, a CMV / MYO7A chimeric promoter, or alternative promoters. The retroviral vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0548] In another embodiment, the retroviral vectors of the present invention can be used to introduce the corrective ATP-binding cassette gene ABCA4 (also known as ABCR) into photoreceptor cells, thereby attenuating or reversing the pathophysiology leading to Stargardt disease. The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or alternative viral envelope proteins. The gene carried by the retroviral vector is the ABCA4 cDNA encoding the ABCA4 protein. Expression of the ABCA4 gene can be driven by a CMV promoter, a photoreceptor-specific promoter, such as rhodopsin kinase, or an alternative promoter. The retroviral vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0549] In another embodiment, the retroviral vectors of the present invention can be used as gene therapy products designed to prevent the recurrence of abnormal blood vessel growth and / or vascular leakage in the eyes of patients with wet age-related macular degeneration (AMD), diabetic macular edema, or retinal vein occlusion, and / or to prevent abnormal blood vessel growth in the eyes of patients with dry age-related macular degeneration (AMD). The retroviral vector delivers one or more genes encoding one or more anti-angiogenic proteins, such as angiostatin and / or endostatin. The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or alternative viral envelope proteins. In one embodiment, the retroviral vector expresses the human endostatin and angiostatin genes in a bicistronic configuration utilizing an internal ribosome entry site (IRES) for delivery to retinal pigment epithelial cells. Expression of the anti-angiogenic gene(s) can be driven by a CMV, RPE-specific promoter, such as the vitelliform macular dystrophy 2 (VMD2) promoter (more recently known as the bestrophin promoter), or by an alternative promoter. The retroviral vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0550] In another embodiment, the retroviral vectors of the present invention can be used as gene therapy products designed to prevent the recurrence of abnormal vascular growth in the eyes of patients with wet age-related macular degeneration (AMD). The retroviral vector delivers one or more genes encoding one or more antiangiogenic proteins, such as angiostatin and / or endostatin. The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or alternative viral envelope proteins. In one embodiment, the retroviral vector utilizes an internal ribosome entry site (IRES) to express the human endostatin and angiostatin genes in a bicistronic configuration for delivery to retinal pigment epithelial cells. Expression of the antiangiogenic gene(s) can be driven by a CMV, RPE-specific promoter, such as the vitelloid macular dystrophy 2 (VMD2) promoter (more recently known as the bestrophin promoter), or by an alternative promoter. Retroviral vectors can be administered by direct subretinal injection after vitrectomy of the eye.

[0551] In another embodiment, the retroviral vectors of the present invention can be used as gene therapy products designed to prevent corneal graft rejection as a result of angiogenesis by delivering antiangiogenic gene(s) to the donor cornea before transplantation. The retroviral vector is a non-replicating, self-inactivating, minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G, Ebola, or alternative viral envelope proteins. In one embodiment, the retroviral vector expresses antiangiogenic gene(s), such as the human endostatin and angiostatin genes in a bicistronic configuration, utilizing an internal ribosome entry site (IRES) for ex vivo delivery to the corneal graft. The retroviral vector can be applied to corneal graft tissue ex vivo, and the transduced donor tissue can also be preserved prior to transplantation. Expression of the antiangiogenic gene(s) can be driven by a constitutive promoter, such as the CMV promoter. However, it is possible that alternative promoters can be used.

[0552] In another embodiment, the retroviral vector of the present invention can be used as a gene therapy designed to prevent the recurrence of abnormal blood vessel growth and / or vascular leakage in the eyes of patients with wet age-related macular degeneration (AMD), diabetic macular edema, or retinal vein occlusion, and / or to prevent abnormal blood vessel growth in the eyes of patients with dry age-related macular degeneration (AMD). This retroviral vector delivers a gene encoding a soluble form of fms-like tyrosine kinase. (Soluble Flt-1) retroviral vectors are non-replicating, self-inactivating minimal lentiviral vectors derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV) that can be pseudotyped with VSV-G or alternative viral envelope proteins. Expression of the soluble Flt-1 gene can be driven by a CMV, RPE-specific promoter, such as the vitelloid macular dystrophy 2 (VMD2) promoter (more recently known as the bestrophin promoter), or by an alternative promoter. Retroviral vectors can be administered by direct subretinal injection after vitrectomy of the eye.

[0553] In another embodiment, the retroviral vectors of the present invention can be used as gene therapy products designed to prevent the recurrence of abnormal blood vessel growth and / or vascular leakage in the eyes of patients with wet age-related macular degeneration (AMD), diabetic macular edema, or retinal vein occlusion, and / or to prevent abnormal blood vessel growth in the eyes of patients with dry age-related macular degeneration (AMD). The retroviral vector delivers one or more genes encoding pigment epithelium-derived factor protein (PEDF). The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or alternative viral envelope proteins. Expression of the PEDF gene can be driven by an RPE-specific promoter, such as CMV, the vitelloid macular dystrophy 2 (VMD2) promoter (more recently known as the bestrophin promoter), or by an alternative promoter. The retroviral vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0554] In another embodiment, the retroviral vector of the present invention can be used as a gene therapy product designed to prevent the recurrence of abnormal blood vessel growth and / or vascular leakage in the eyes of patients with wet age-related macular degeneration (AMD), diabetic macular edema, or retinal vein occlusion, and / or to prevent abnormal blood vessel growth in the eyes of patients with dry age-related macular degeneration (AMD). The retroviral vector delivers one or more genes encoding a vascular endothelial growth factor (VEGF) inhibitor, such as an anti-VEGF antibody or its binding fragment (e.g., aflibercept), a VEGF-specific aptamer, or a soluble form of a VEGF receptor, including, but not limited to, a VEGF-blocking peptide or polypeptide, and / or a platelet-derived growth factor (PDGF) inhibitor, such as an anti-PDGF antibody or its binding fragment, a PDGF-specific aptamer, or a soluble form of a PDGF receptor, including, but not limited to, a PDGF-blocking peptide or polypeptide. Retroviral vectors are non-replicating, self-inactivating minimal lentiviral vectors derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or alternative viral envelope proteins. In one embodiment, the retroviral vector expresses a bicistronic VEGF inhibitor and a PDGF inhibitor using an internal ribosome entry site (IRES) for delivery to retinal pigment epithelial cells. Expression of the gene(s) can be driven by an RPE-specific promoter, such as CMV, the vitelloid macular dystrophy 2 (VMD2) promoter (more recently known as the bestrophin promoter), or by an alternative promoter. Retroviral vectors can be administered by direct subretinal injection after vitrectomy of the eye.

[0555] In another embodiment, the retroviral vectors of the present invention can be used to introduce a cassette encoding the corrective gene vitelliform macular dystrophy 2 (VMD2) and a microRNA (miRNA) specific for a disease-associated form of VMD2, or a cassette encoding a corrective RDS gene encoding peripherin 2 and a miRNA specific for a disease-associated form of RDS, into retinal pigment epithelial cells, thereby attenuating or reversing the pathophysiology leading to Best disease or Best vitelliform macular degeneration (BVMD). The retroviral vectors are non-replicating, self-inactivating minimal lentiviral vectors derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or alternative viral envelope proteins. Gene expression can be driven by an RPE-specific promoter, such as the CMV vitelliform macular dystrophy 2 (VMD2) promoter (more recently known as the bestrophin promoter), or by an alternative promoter. Retroviral vectors can be administered by direct subretinal injection after vitrectomy of the eye.

[0556] In another embodiment, the retroviral vector of the present invention can be used to introduce the corrective retinaldehyde-binding protein 1 (RLBP1) gene into retinal pigment epithelial cells, thereby attenuating or reversing the pathophysiology leading to RLBP1-associated retinal dystrophy. The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or alternative viral envelope proteins. The gene carried by the retroviral vector is the RLBP1 cDNA encoding the RLBP1 protein. Expression of the RLBP1 gene can be driven by an RPE-specific promoter, such as the CMV vitelliform macular dystrophy 2 (VMD2) promoter (more recently known as the bestrophin promoter), or by an alternative promoter. The retroviral vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0557] In another embodiment, the retroviral vector of the present invention can be used as a gene therapy product designed to treat glaucoma. The retroviral vector delivers one or more genes encoding COX-2 and / or prostaglandin F2α receptor (FPR), which act to reduce intraocular pressure. The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or alternative viral envelope proteins. In one embodiment, the retroviral vector expresses a bicistronic COX-2 and prostaglandin F2α receptor (FPR) gene using an internal ribosome entry site (IRES) for delivery to the anterior chamber of the eye. Expression of the gene(s) can be driven by CMV or alternative promoters. The retroviral vector can be administered by transcorneal injection.

[0558] In another embodiment, the retroviral vector of the present invention can be used to introduce a corrective harmonin gene to attenuate or reverse the pathophysiology leading to Usher syndrome 1c. The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or an alternative viral envelope protein. The gene carried by the retroviral vector is harmonin cDNA, which encodes the harmonin protein. Expression of the harmonin gene can be driven by the CMV promoter or an alternative promoter. The retroviral vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0559] In another embodiment, the retroviral vectors of the present invention can be used to introduce the corrective Rab escort protein 1 (REP1) gene to attenuate or reverse the pathophysiology leading to total choroidal atrophy. The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or alternative viral envelope proteins. The gene carried by the retroviral vector is the REP1 cDNA encoding the REP1 protein. Expression of the REP1 gene can be driven by the CMV promoter or an alternative promoter. The retroviral vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0560] In another embodiment, the retroviral vectors of the present invention can be used to introduce corrective cyclic nucleotide-gated channel beta 2 (CNGB2) and / or cyclic nucleotide-gated channel alpha 3 (CNGA3) genes into the eye to attenuate or reverse the pathophysiology leading to color vision deficiency. The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or an alternative viral envelope protein. The gene(s) carried by the retroviral vector are CNGB2 and / or CNGA3 genes encoding CNGB2 and / or CNGA3 proteins. Expression of the gene(s) can be driven by a CMV promoter or an alternative promoter. The retroviral vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0561] In another embodiment, the retroviral vector of the present invention can be used to introduce a corrective CEP290 gene into the eye to attenuate or reverse the pathophysiology leading to Leber congenital amaurosis (LCA). The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or alternative viral envelope proteins. The gene carried by the retroviral vector is the CEP290 gene, which encodes a 290 kDa centrosomal protein. Expression of the CEP290 gene can be driven by the CMV promoter or an alternative promoter. The retroviral vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0562] In another embodiment, the retroviral vector of the present invention can be used to introduce the corrective retinitis pigmentosa GTPase regulator (RPGR) gene into the eye to attenuate or reverse the pathophysiology leading to X-linked retinitis pigmentosa. The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or alternative viral envelope proteins. The gene carried by the retroviral vector is the RPGR cDNA encoding the RPGR protein. Expression of the RPGR gene can be driven by the CMV promoter or alternative promoters. The retroviral vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0563] In another embodiment, the retroviral vectors of the present invention can be used to introduce a corrective retinoschisin 1 (RS1) gene into the eye to attenuate or reverse the pathophysiology leading to X-linked retinochisis. The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or alternative viral envelope proteins. The gene carried by the retroviral vector is RS1 cDNA, which encodes the RS1 protein. Expression of the RS1 gene can be driven by the CMV promoter or an alternative promoter. The retroviral vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0564] In another embodiment, the retroviral vectors of the present invention can be used to introduce the corrective retinitis pigmentosa 1 (RP1) gene into the eye to attenuate or reverse the pathophysiology leading to retinitis pigmentosa. The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or alternative viral envelope proteins. The gene carried by the retroviral vector is the RP1 cDNA encoding the RP1 protein. Expression of the RP1 gene can be driven by a CMV promoter, a photoreceptor-specific promoter, such as rhodopsin kinase, or an alternative promoter. The retroviral vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0565] In another embodiment, the retroviral vectors of the present invention can be used to introduce a corrective retinal pigment epithelium-specific 65 kDa protein (RPE65) gene to attenuate or reverse the pathophysiology leading to Leber congenital amaurosis (LCA) type 2. The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or alternative viral envelope proteins. The gene carried by the retroviral vector is the RPE65 cDNA encoding the RPE65 protein. Expression of the RPE65 gene can be driven by an RPE-specific promoter, such as CMV, the vitelliform macular dystrophy 2 (VMD2) promoter (more recently known as the bestrophin promoter), or by an alternative promoter. The retroviral vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0566] In another embodiment, the retroviral vector of the present invention can be used to introduce the corrective human proline / arginine-rich terminal leucine-rich repeat protein (PRELP) gene to attenuate or reverse the pathophysiology leading to wet age-related macular degeneration (AMD), dry AMD, diabetic macular edema, or retinal vein occlusion. The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or alternative viral envelope proteins. The gene carried by the retroviral vector is the PRELP cDNA encoding the PRELP protein. Expression of the PRELP gene can be driven by an RPE-specific promoter, such as CMV, the vitelloid macular dystrophy 2 (VMD2) promoter (more recently known as the bestrophin promoter), or by an alternative promoter. The retroviral vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0567] In another embodiment, the retroviral vector of the present invention can be used to introduce a nucleic acid sequence encoding a synthetic myocilin-specific miRNA into the eye to attenuate or reverse the pathophysiology leading to juvenile open-angle glaucoma by knocking down myocilin expression. The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or an alternative viral envelope protein. Expression of the synthetic myocilin-specific miRNA can be driven by a CMV promoter or an alternative promoter. The retroviral vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0568] In another embodiment, the retroviral vectors of the present invention can be used to introduce nucleic acid sequences encoding glutamate-cysteine ​​ligase (GCL) and / or glutathione synthetase (GSS), rate-limiting enzymes in the glutathione biosynthetic pathway, and / or synthetic gamma-glutamyltransferase (GGT)-specific miRNAs into the eye to attenuate or reverse the pathophysiology leading to retinitis pigmentosa by gene augmentation and / or knockdown. The retroviral vector is, for example, a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or an alternative viral envelope protein. Expression of the GCL and / or GSS gene(s) and / or synthetic GGT-specific miRNA can be driven by a CMV promoter or an alternative promoter. In one embodiment, the retroviral vector can utilize one or more internal ribosome entry sites (IRES) to express a multicistronic gene(s) and / or synthetic miRNA. The retroviral vector can be administered by direct delivery to the anterior chamber of the eye.

[0569] In another embodiment, the retroviral vectors of the present invention can be used as gene therapy products designed to treat neurodegenerative disorders such as frontotemporal dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, and motor neuron disorders such as amyotrophic lateral sclerosis (ALS). 145 , VEGF 165 or VEGF 189 The retroviral vector delivers a gene encoding a VEGF protein, which may be a VEGF-A isoform such as VEGF-A, or may be VEGF-B, VEGF-C, or VEGF-D, and such genes have neuroprotective effects. The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which may be pseudotyped with rabies G, VSV-G, or alternative viral envelope proteins. Gene expression can be driven by a CMV or alternative promoter. Retroviral vectors can be administered by direct injection into large muscles or directly into the cerebrospinal fluid via intrathecal or intracerebroventricular injection.

[0570] In another embodiment, the retroviral vector of the present invention can be used as a gene therapy product designed to treat cystic fibrosis. This retroviral vector delivers a gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with influenza hemagglutinin, Sendai virus envelope F or HN, Ebola, baculovirus GP64, or alternative viral envelope proteins. Gene expression can be driven by CMV or alternative promoters. Retroviral vectors can be administered intranasally, by use of a nebulizer, or by direct delivery to the lungs via bronchoalveolar lavage.

[0571] In another embodiment, the retroviral vectors of the present invention can be used to introduce corrective N-sulfoglucosamine sulfohydrolase (SGSH) and / or sulfatase modifier 1 (SUMF1) genes into the brain to attenuate or reverse the pathophysiology leading to Sanfilippo syndrome A. The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or alternative viral envelope proteins. The gene(s) carried by the retroviral vector are SGSH cDNA encoding the SGSH protein and / or the SUMF1 gene encoding the SUMF1 protein. Expression of the gene(s) can be driven by a CMV promoter or an alternative promoter. In one embodiment, the retroviral vector can utilize an internal ribosome entry site (IRES) to express the SGSH and SUMF1 genes in a bicistronic configuration. The retroviral vector can be administered by direct intracerebral injection.

[0572] In another embodiment, the retroviral vectors of the present invention can be used to introduce a corrected acid alpha-glycosidase (GAA) gene into large muscles and / or the lungs to attenuate or reverse the pathophysiology leading to Pompe disease. The retroviral vector delivers a gene encoding the GAA protein. The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with influenza hemagglutinin, Sendai virus envelope F or HN, Ebola, baculovirus GP64, rabies G, VSV-G, or alternative viral envelope proteins. Gene expression can be driven by a CMV or alternative promoter. The retroviral vector can be administered (i) by direct injection into large muscles and / or (ii) by intranasal, nebulizer, or direct delivery to the lungs via bronchoalveolar lavage.

[0573] In another embodiment, the retroviral vector of the present invention can be used ex vivo to transduce autologous or allogeneic T cells with a nucleic acid sequence encoding a CD19-specific chimeric antigen receptor (CAR19).These transduced T cells are then infused into a subject to treat CD19-expressing cancers and leukemias.The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or alternative viral envelope proteins.The expression of the nucleic acid sequence encoding the CAR can be driven by EF1α, CMV, or alternative promoters.

[0574] In another embodiment, the retroviral vector of the present invention can be used ex vivo to transduce autologous or allogeneic T cells with a nucleic acid sequence encoding a 5T4-specific chimeric antigen receptor (CAR).These transduced T cells are then infused into a subject to treat 5T4-expressing cancers and leukemias.The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or alternative viral envelope proteins.The expression of the nucleic acid sequence encoding the 5T4 CAR can be driven by EF1α, CMV, or alternative promoters.

[0575] As known to those skilled in the art, chimeric antigen receptors (CARs) specific to a range of cancer- or leukemia-associated polypeptides can be produced. The retroviral vectors of the present invention can be used ex vivo to transduce autologous or allogeneic T cells with a nucleic acid sequence encoding a chimeric antigen receptor (CAR) specific to any cancer- or leukemia-associated polypeptide. These transduced T cells are then infused into a subject to treat cancers and leukemias that express the cancer- or leukemia-associated polypeptide to which the CAR binds. Retroviral vectors are non-replicating, self-inactivating minimal lentiviral vectors derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or alternative viral envelope proteins. Expression of the nucleic acid sequence encoding the CAR can be driven by EF1α, CMV, or alternative promoters. Suitable cancer or leukemia-associated polypeptides that can be targeted by the CAR include, but are not limited to, mesothelin, folate receptor alpha, immunoglobulin kappa light chain, CD30, carcinoembryonic antigen (CEA), CD138, ganglioside G2 (GD2), CD33, CD22, epidermal growth factor receptor (EGFR) such as EGF VIII, IL-13Rα2, CD20, ErbB such as Her2, prostate-specific membrane antigen (PSMA), Lewis Y antigen, and fibroblast activation protein (FAB).

[0576] In another embodiment, the retroviral vectors of the present invention can be used ex vivo to transduce autologous or allogeneic T cells with nucleic acid sequences encoding T cell receptors (TCRs) specific for peptide-MHC expressed on diseased, leukemia, or cancerous cells. These transfected T cells are then infused into a subject to treat diseases, cancers, or leukemias associated with the expression of TCR-binding peptide-MHC. The retroviral vectors are non-replicating, self-inactivating, minimal lentiviral vectors derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or alternative viral envelope proteins. Expression of the nucleic acid sequence encoding the TCR can be driven by EF1α, CMV, or alternative promoters. The TCRs encoded by the vectors of the present invention can be single-chain TCRs (scTCRs) or dimeric TCRs (dTCRs). As known to those skilled in the art, suitable dTCRs include those described in WO 2003 / 020763, and suitable scTCRs include those described in WO 1999 / 018129. In certain aspects of this embodiment, TCR-transfected T cells may be used to treat AIDS, leukemia, and cancers, including myeloma and sarcoma.

[0577] In another embodiment, the retroviral vectors of the present invention can be used to introduce a gene encoding the common gamma chain (CD132) to treat X-linked severe combined immunodeficiency (SCID). The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or an alternative viral envelope protein. Gene expression can be driven by the CMV promoter or an alternative promoter. The retroviral vectors of the present invention can be used ex vivo to transduce bone marrow stem cells. These transduced bone marrow stem cells can then be infused into a subject to treat the disease.

[0578] In another embodiment, the retroviral vectors of the present invention can be used to introduce a gene encoding adenosine deaminase to treat severe combined immunodeficiency syndrome (ADA) (SCID). The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or alternative viral envelope proteins. Gene expression can be driven by a CMV promoter or an alternative promoter. The retroviral vectors of the present invention can be used ex vivo to transduce bone marrow stem cells. These transduced bone marrow stem cells can then be infused into a subject to treat the disease.

[0579] In another embodiment, the retroviral vectors of the present invention can be used to treat Wiskott-Aldrich syndrome (WAS) by introducing a gene encoding the WAS protein. The retroviral vector is a non-replicating, self-inactivating, minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or alternative viral envelope proteins. Gene expression can be driven by the CMV promoter or alternative promoters. The retroviral vectors of the present invention can be used ex vivo to transduce bone marrow stem cells. These transduced bone marrow stem cells can then be infused into a subject to treat the disease.

[0580] In another embodiment, the retroviral vectors of the present invention can be used to treat sickle cell disease or thalassemia by introducing a gene encoding one of several globins, including wild-type β-globin, wild-type fetal globin, and mutated "anti-sickling" globin. As known to those skilled in the art, examples of anti-sickling globins include, but are not limited to, those described in International Publication Nos. WO 2014 / 043131 and WO 1996 / 009385. The retroviral vector is a non-replicating, self-inactivating, minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or an alternative viral envelope protein. Gene expression can be driven by a CMV promoter or an alternative promoter. The retroviral vectors of the present invention can be used ex vivo to transduce bone marrow stem cells. These transduced bone marrow stem cells can then be infused into a subject to treat the disease.

[0581] In another embodiment, the retroviral vector of the present invention can be used to introduce a corrective gene, Factor VIII, into liver, muscle, or fat cells to treat hemophilia A. The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or an alternative viral envelope protein. The gene carried by the retroviral vector is Factor VIII. Expression of the Factor VIII gene can be driven by the CMV promoter or an alternative promoter.

[0582] In another embodiment, the retroviral vector of the present invention can be used to introduce a corrective gene, Factor IX, into liver, muscle, or fat cells to treat hemophilia B. The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or an alternative viral envelope protein. The gene carried by the retroviral vector is Factor IX. Expression of the Factor IX gene can be driven by the CMV promoter or an alternative promoter.

[0583] In another embodiment, the retroviral vector of the present invention can be used to treat Fabry disease by introducing a gene encoding alpha-galactosidase A (α-GAL A). The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or an alternative viral envelope protein. The gene carried by the retroviral vector is the GLA cDNA, which encodes the α-GAL A protein. Expression of the gene can be driven by the CMV promoter or an alternative promoter. The retroviral vector of the present invention can also be used to transduce the hematopoietic CD34 + These transduced hematopoietic CD34 cells can then be used ex vivo to transduce stem cells. + The stem cells can be injected into a subject to treat a disease.

[0584] In another embodiment, the retroviral vectors of the present invention can be used to introduce a gene encoding a defective enzyme to treat a form of porphyria. The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or an alternative viral envelope protein. The gene carried by the retroviral vector is a gene encoding a defective enzyme associated with the type of porphyria to be treated, selected from the table below. Expression of the gene can be driven by the CMV promoter or an alternative promoter.

[0585] [Table 1]

[0586] In another embodiment, the retroviral vectors of the present invention can be used to introduce a gene encoding a defective enzyme to treat a form of mucopolysaccharidosis. The retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV), which can be pseudotyped with VSV-G or an alternative viral envelope protein. The gene carried by the retroviral vector is a gene encoding a defective enzyme associated with the type of mucopolysaccharidosis to be treated, selected from the table below. Expression of the gene can be driven by the CMV promoter or an alternative promoter.

[0587] [Table 2]

[0588] Construction of retroviral therapeutic vectors The retroviral vector of the present invention comprises four plasmids: (1) a recombinant retroviral vector genome plasmid encoding the desired transgene(s) and a nucleic acid sequence of the invention; (2) synthetic retroviral gag / pol expression plasmid; (3) an envelope (env) expression plasmid capable of expressing, for example, VSV-G; and (4) RNA-binding protein expression plasmid It can be produced by transient transfection of HEK293T cells with

[0589] Alternatively, a retroviral vector of the invention, such as HIV, may comprise five plasmids: (1) a recombinant HIV vector genome plasmid encoding the required transgene(s), the nucleic acid sequence of the invention, and the RRE sequence; (2) synthetic gag / pol expression plasmid; (3) an envelope (env) expression plasmid capable of expressing, for example, VSV-G; (4) RNA-binding protein expression plasmid, and (5) REV expression plasmid It can be produced by transient transfection of HEK293T cells with

[0590] Alternatively, retroviral vectors of the present invention, such as HIV, can be generated by transient transfection of HEK293T cells with at least one modular construct encoding the components required for the production of a viral vector and TRAP, wherein the viral genome comprises the nucleic acid sequence of the present invention. Suitable modular constructs include, but are not limited to, those described in EP 3502260.

[0591] Alternatively, a transient transfection system may utilize a cell line that stably expresses TRAP.

[0592] Alternatively, the retroviral vectors of the present invention can be produced by using packaging cells that stably express (1) gag / pol, (2) env, and (3) TRAP, and in the case of HIV vectors, Rev, and a plasmid encoding the required transgene(s) and a recombinant retroviral vector genome encoding the nucleic acid sequence of the present invention, which in the case of HIV vectors includes an RRE sequence, is introduced into such cells by transient transfection.

[0593] Alternatively, the retroviral vectors of the invention can be produced in producer cells that stably express a recombinant EIAV vector genome encoding (1) gag / pol, (2) env, (3) TRAP, (4) the required transgene(s) and a nucleic acid sequence of the invention.

[0594] Alternatively, the HIV vectors of the present invention can be produced in producer cells stably expressing (1) gag / pol, (2) env, (3) TRAP, (4) a recombinant HIV vector genome encoding the required transgene(s), the nucleic acid sequence of the present invention and the RRE sequence, and (5) REV.

[0595] AAV therapeutic vectors In another embodiment, the AAV vector of the present invention can be used to introduce three genes encoding three enzymes in the dopamine synthesis pathway to treat Parkinson's disease. The genes carried by the AAV vector can include a truncated human tyrosine hydroxylase (TH*) gene (lacking the N-terminal 160 amino acids involved in the feedback regulation of TH), human aromatic L-amino acid decarboxylase (AADC), and human GTP-cyclohydrolase 1 (CH1) genes. The three enzymes can be encoded by the AAV vector in three separate open reading frames. Alternatively, the AAV vector can encode a fusion of the TH and CH1 enzymes in one open reading frame with the AADC enzyme in the second open reading frame. Gene expression can be driven by a CMV promoter, and the expression cassette can contain one or more IRES elements. The AAV vector can be administered by direct injection into the striatum of the brain.

[0596] In another embodiment, the AAV vectors of the present invention can be used as gene therapy products designed to introduce a corrective MYO7A gene into photoreceptors and their supporting retinal pigment epithelial (RPE) cells, thereby attenuating or reversing the vision loss associated with Usher 1B syndrome. The gene carried by the AAV vector is MYO7A cDNA (a large gene over 100 mb in length) encoding the MYO7A protein. Expression of the large MYO7A gene can be driven by a CMV promoter, a CMV / MYO7A chimeric promoter, or an alternative promoter. The AAV vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0597] In another embodiment, the AAV vector of the present invention can be used to introduce the corrective ATP-binding cassette gene ABCA4 (also known as ABCR) into photoreceptor cells, thereby attenuating or reversing the pathophysiology that leads to Stargardt disease. The gene carried by the AAV vector is the ABCA4 cDNA encoding the ABCA4 protein. Expression of the ABCA4 gene can be driven by a CMV promoter, a photoreceptor-specific promoter such as rhodopsin kinase, or an alternative promoter. The AAV vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0598] In another embodiment, the AAV vector of the present invention can be used as a gene therapy product designed to prevent the recurrence of abnormal blood vessel growth and / or vascular leakage in the eyes of patients with wet age-related macular degeneration (AMD), diabetic macular edema, or retinal vein occlusion, and / or to prevent abnormal blood vessel growth in the eyes of patients with dry age-related macular degeneration (AMD). The AAV vector delivers one or more genes encoding one or more antiangiogenic proteins, such as angiostatin and / or endostatin. In one embodiment, the AAV vector utilizes an internal ribosome entry site (IRES) to express the human endostatin and angiostatin genes in a bicistronic configuration for delivery to retinal pigment epithelial cells. Expression of the antiangiogenic gene(s) can be driven by a CMV, RPE-specific promoter, such as the vitelliform macular dystrophy 2 (VMD2) promoter (more recently known as the bestrophin promoter), or by an alternative promoter. The AAV vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0599] In another embodiment, the AAV vector of the present invention can be used as a gene therapy product designed to prevent corneal graft rejection as a result of angiogenesis by delivering antiangiogenic gene(s) to the donor cornea before transplantation. In one embodiment, the AAV vector expresses antiangiogenic gene(s), such as the human endostatin and angiostatin genes in a bicistronic configuration, using an internal ribosome entry site (IRES) for ex vivo delivery to the corneal graft. The AAV vector can be applied to the corneal graft tissue ex vivo, and the transduced donor tissue can also be preserved before transplantation. Expression of the antiangiogenic gene(s) can be driven by a constitutive promoter, such as a CMV promoter. However, it is also possible that alternative promoters can be used.

[0600] In another embodiment, the AAV vector of the present invention can be used as a gene therapy designed to prevent the recurrence of abnormal blood vessel growth and / or vascular leakage in the eyes of patients with wet age-related macular degeneration (AMD), diabetic macular edema, or retinal vein occlusion, and / or to prevent abnormal blood vessel growth in the eyes of patients with dry age-related macular degeneration (AMD). This AAV vector delivers a gene encoding a soluble form of fms-like tyrosine kinase (soluble Flt-1). Expression of the soluble Flt-1 gene can be driven by a CMV, RPE-specific promoter, such as the vitelliform macular dystrophy 2 (VMD2) promoter (more recently known as the bestrophin promoter), or by an alternative promoter. The AAV vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0601] In another embodiment, the AAV vector of the present invention can be used as a gene therapy product designed to prevent the recurrence of abnormal blood vessel growth and / or vascular leakage in the eyes of patients with wet age-related macular degeneration (AMD), diabetic macular edema, or retinal vein occlusion, and / or to prevent abnormal blood vessel growth in the eyes of patients with dry age-related macular degeneration (AMD). This AAV vector delivers one or more genes encoding pigment epithelium-derived factor protein (PEDF). Expression of the PEDF gene can be driven by an RPE-specific promoter, such as CMV, vitelliform macular dystrophy 2 (VMD2) promoter (more recently known as the bestrophin promoter), or by an alternative promoter. The AAV vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0602] In another embodiment, the AAV vectors of the present invention can be used as gene therapy products designed to prevent the recurrence of abnormal blood vessel growth and / or vascular leakage in the eyes of patients with wet age-related macular degeneration (AMD), diabetic macular edema, or retinal vein occlusion, and / or to prevent abnormal blood vessel growth in the eyes of patients with dry age-related macular degeneration (AMD). The AAV vector delivers one or more genes encoding a vascular endothelial growth factor (VEGF) inhibitor, such as a VEGF-blocking peptide or polypeptide, including, but not limited to, an anti-VEGF antibody or a binding fragment thereof (e.g., aflibercept), a VEGF-specific aptamer, or a soluble form of the VEGF receptor, and / or a platelet-derived growth factor (PDGF) inhibitor, such as a PDGF-blocking peptide or polypeptide, including, but not limited to, an anti-PDGF antibody or a binding fragment thereof, a PDGF-specific aptamer, or a soluble form of the PDGF receptor. In one embodiment, the AAV vector expresses a bicistronic VEGF inhibitor and a PDGF inhibitor using an internal ribosome entry site (IRES) for delivery to retinal pigment epithelial cells. The expression of the gene(s) can be driven by an RPE-specific promoter, such as CMV, vitelliform macular dystrophy 2 (VMD2) promoter (more recently known as the bestrophin promoter), or by an alternative promoter. The AAV vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0603] In another embodiment, the AAV vectors of the present invention are used to introduce a cassette encoding the corrective gene vitelliform macular dystrophy 2 (VMD2) and a microRNA (miRNA) specific for a disease-associated form of VMD2, or a cassette encoding the corrective RDS gene encoding peripherin 2 and a miRNA specific for a disease-associated form of RDS, into retinal pigment epithelial cells, thereby attenuating or reversing the pathophysiology leading to Best disease or Best vitelliform macular degeneration (BVMD). Expression of the gene can be driven by an RPE-specific promoter, such as the CMV vitelliform macular dystrophy 2 (VMD2) promoter (more recently known as the bestrophin promoter), or by an alternative promoter. The AAV vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0604] In another embodiment, the AAV vector of the present invention can be used to introduce the corrective retinaldehyde-binding protein 1 gene (RLBP1) into retinal pigment epithelial cells, thereby attenuating or reversing the pathophysiology that leads to RLBP1-associated retinal dystrophy. The gene carried by the AAV vector is the RLBP1 cDNA encoding the RLBP1 protein. The expression of the RLBP1 gene can be driven by an RPE-specific promoter, such as CMV, vitelliform macular dystrophy 2 (VMD2) promoter (more recently known as the bestrophin promoter), or by an alternative promoter. The AAV vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0605] In another embodiment, the AAV vector of the present invention can be used as a gene therapy product designed to treat glaucoma. This AAV vector delivers one or more genes encoding COX-2 and / or prostaglandin F2α receptor (FPR). In one embodiment, the AAV vector expresses the bicistronic COX-2 and prostaglandin F2α receptor (FPR) genes using an internal ribosome entry site (IRES) for delivery to the anterior chamber of the eye. Expression of the gene(s) can be driven by a CMV or alternative promoter. The AAV vector can be administered via transcorneal injection.

[0606] In another embodiment, the AAV vector of the present invention can be used to introduce a corrective harmonin gene to attenuate or reverse the pathophysiology that leads to Usher syndrome 1c. The gene carried by the AAV vector is harmonin cDNA encoding the harmonin protein. Expression of the harmonin gene can be driven by a CMV promoter or an alternative promoter. The AAV vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0607] In another embodiment, the AAV vector of the present invention can be used to introduce the corrective Rab escort protein 1 (REP1) gene to attenuate or reverse the pathophysiology that leads to total choroidal atrophy. The gene carried by the AAV vector is the REP1 cDNA encoding the REP1 protein. Expression of the REP1 gene can be driven by a CMV promoter or an alternative promoter. The AAV vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0608] In another embodiment, the AAV vector of the present invention can be used to introduce corrective cyclic nucleotide-gated channel beta 2 (CNGB2) and / or cyclic nucleotide-gated channel alpha 3 (CNGA3) genes into the eye to attenuate or reverse the pathophysiology that leads to color vision deficiency. The gene(s) carried by the AAV vector are the CNGB2 and / or CNGA3 genes encoding CNGB2 and / or CNGA3 proteins. The expression of the gene(s) can be driven by a CMV promoter or an alternative promoter. The AAV vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0609] In another embodiment, the AAV vector of the present invention can be used to introduce a corrective CEP290 gene into the eye to attenuate or reverse the pathophysiology that leads to Leber congenital amaurosis (LCA). The gene carried by the AAV vector is the CEP290 gene, which encodes a 290 kDa centrosomal protein. The expression of the CEP290 gene can be driven by a CMV promoter or an alternative promoter. The AAV vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0610] In another embodiment, the AAV vector of the present invention can be used to introduce the corrective retinitis pigmentosa GTPase regulator (RPGR) gene into the eye to attenuate or reverse the pathophysiology that leads to X-linked retinitis pigmentosa. The gene carried by the AAV vector is the RPGR cDNA encoding the RPGR protein. The expression of the RPGR gene can be driven by a CMV promoter or an alternative promoter. The AAV vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0611] In another embodiment, the AAV vector of the present invention can be used to introduce the corrective retinoschisin 1 (RS1) gene into the eye to attenuate or reverse the pathophysiology leading to X-linked retinochisis. The gene carried by the AAV vector is RS1 cDNA encoding the RS1 protein. Expression of the RS1 gene can be driven by a CMV promoter or an alternative promoter. The AAV vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0612] In another embodiment, the AAV vector of the present invention can be used to introduce the corrective retinitis pigmentosa 1 (RP1) gene into the eye to attenuate or reverse the pathophysiology that leads to retinitis pigmentosa. The gene carried by the AAV vector is the RP1 cDNA encoding the RP1 protein. The expression of the RP1 gene can be driven by a CMV promoter, a photoreceptor-specific promoter, such as rhodopsin kinase, or an alternative promoter. The AAV vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0613] In another embodiment, the AAV vector of the present invention can be used to introduce the corrective retinal pigment epithelium-specific 65 kDa protein (RPE65) gene to attenuate or reverse the pathophysiology leading to Leber congenital amaurosis (LCA) type 2. The gene carried by the AAV vector is the RPE65 cDNA encoding the RPE65 protein. Expression of the RPE65 gene can be driven by an RPE-specific promoter, such as CMV, the vitelliform macular dystrophy 2 (VMD2) promoter (more recently known as the bestrophin promoter), or by an alternative promoter. The AAV vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0614] In another embodiment, the AAV vector of the present invention can be used to introduce the corrective human proline / arginine-rich terminal leucine-rich repeat protein (PRELP) gene to attenuate or reverse the pathophysiology leading to wet age-related macular degeneration (AMD), dry AMD, diabetic macular edema, or retinal vein occlusion. The gene carried by the AAV vector is the PRELP cDNA encoding the PRELP protein. Expression of the PRELP gene can be driven by an RPE-specific promoter, such as CMV, the vitelliform macular dystrophy 2 (VMD2) promoter (more recently known as the bestrophin promoter), or by an alternative promoter. The AAV vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0615] In another embodiment, the AAV vector of the present invention can be used to introduce a nucleic acid sequence encoding synthetic myocilin-specific miRNA into the eye, thereby reducing or reversing the pathophysiology of juvenile open-angle glaucoma by knocking down the expression of myocilin.The expression of synthetic myocilin-specific miRNA can be driven by a CMV promoter or an alternative promoter.The AAV vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0616] In another embodiment, the AAV vector of the present invention can be used to introduce nucleic acid sequences encoding glutamate-cysteine ​​ligase (GCL) and / or glutathione synthetase (GSS), which are rate-limiting enzymes in the glutathione biosynthesis pathway, and / or synthetic gamma-glutamyltransferase (GGT)-specific miRNAs into the eye to attenuate or reverse the pathophysiology leading to retinitis pigmentosa by gene augmentation and / or knockdown. Expression of the GCL and / or GSS gene(s) and / or synthetic GGT-specific miRNA can be driven by a CMV promoter or an alternative promoter. In one embodiment, the AAV vector can express a multicistronic gene(s) and / or synthetic miRNA using one or more internal ribosome entry sites (IRES). The AAV vector can be administered by direct delivery to the anterior chamber of the eye.

[0617] In another embodiment, the AAV vectors of the present invention can be used as gene therapy products designed to treat neurodegenerative disorders such as frontotemporal dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, and motor neuron disorders such as amyotrophic lateral sclerosis (ALS). 145 , VEGF 165 or VEGF 189 The gene encoding VEGF protein can be VEGF-A isoform such as VEGF-A, or VEGF-B, VEGF-C or VEGF-D, and this gene has neuroprotective effect.The expression of gene can be driven by CMV or alternative promoter.AAV vector can be administered by direct injection into large muscle groups, or by direct injection into cerebrospinal fluid by intrathecal or intraventricular injection.

[0618] In another embodiment, the AAV vector of the present invention can be used as a gene therapy product designed to treat cystic fibrosis.This AAV vector delivers the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR).The expression of the gene can be driven by CMV or alternative promoter.The AAV vector can be administered intranasally, by using a nebulizer, or by direct delivery to the lungs via bronchoalveolar lavage fluid.

[0619] In another embodiment, the AAV vector of the present invention can be used to introduce corrective N-sulfoglucosamine sulfohydrolase (SGSH) and / or sulfatase modifier 1 (SUMF1) gene(s) into the brain to attenuate or reverse the pathophysiology leading to Sanfilippo syndrome A. The gene(s) carried by the AAV vector are the SGSH cDNA encoding the SGSH protein and / or the SUMF1 gene encoding the SUMF1 protein. Expression of the gene(s) can be driven by a CMV promoter or an alternative promoter. In one embodiment, the AAV vector can express the SGSH and SUMF1 genes in a bicistronic configuration using an internal ribosome entry site (IRES). The AAV vector can be administered by direct intracerebral injection.

[0620] In another embodiment, the AAV vector of the present invention can be used to introduce a corrected acid alpha-glycosidase (GAA) gene into the large muscles and / or lungs to attenuate or reverse the pathophysiology that leads to Pompe disease. The AAV vector delivers a gene encoding the GAA protein. Gene expression can be driven by a CMV or alternative promoter. The AAV vector can be administered (i) by direct injection into the large muscles and / or (ii) by intranasal, nebulizer, or direct delivery into the lungs via bronchoalveolar lavage fluid.

[0621] In another embodiment, the AAV vectors of the invention can be used to introduce a corrective gene, Factor VIII, into liver, muscle, or fat cells to treat hemophilia A. The gene carried by the AAV vector is Factor VIII. Expression of the Factor VIII gene can be driven by the CMV promoter or an alternative promoter.

[0622] In another embodiment, the AAV vectors of the invention can be used to introduce a corrective gene, Factor IX, into liver, muscle, or fat cells to treat hemophilia B. The gene carried by the AAV vector is Factor IX. Expression of the Factor IX gene can be driven by the CMV promoter or an alternative promoter.

[0623] In another embodiment, the AAV vectors of the present invention can be used to treat a form of porphyria by introducing a gene encoding a defective enzyme. The gene carried by the AAV vector is a gene encoding a defective enzyme associated with the type of porphyria to be treated selected from the table below. Expression of the gene can be driven by a CMV promoter or an alternative promoter.

[0624] [Table 3]

[0625] In another embodiment, the AAV vectors of the present invention can be used to treat a form of mucopolysaccharidosis by introducing a gene encoding a defective enzyme. The gene carried by the AAV vector is a gene encoding a defective enzyme associated with the type of mucopolysaccharidosis to be treated selected from the table below. Expression of the gene can be driven by a CMV promoter or an alternative promoter.

[0626] [Table 4]

[0627] In another embodiment, the AAV vector of the present invention can be used as a gene therapy product designed to prevent the recurrence of abnormal vascular growth in the eyes of patients with wet age-related macular degeneration (AMD). This AAV vector delivers one or more genes encoding one or more anti-angiogenic proteins, such as angiostatin and / or endostatin. In one embodiment, the AAV vector expresses a bicistronic human endostatin and angiostatin gene using an internal ribosome entry site (IRES) for delivery to retinal pigment epithelial cells. Expression of the anti-angiogenic gene(s) can be driven by a CMV, RPE-specific promoter, such as the vitelliform macular dystrophy 2 (VMD2) promoter (more recently known as the bestrophin promoter), or by an alternative promoter. The AAV vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0628] In another embodiment, the AAV vectors of the present invention can be used as gene therapy products designed to treat neurodegenerative disorders such as frontotemporal dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, and motor neuron disorders such as amyotrophic lateral sclerosis (ALS). 145 , VEGF 165 or VEGF 189 The AAV vector can deliver a gene encoding a VEGF protein, which can be a VEGF-A isoform such as VEGF-A, or VEGF-B, VEGF-C, or VEGF-D, and this gene has a neuroprotective effect. The expression of the gene can be driven by a CMV or alternative promoter. The AAV vector can be administered by intraventricular injection or by intrathecal injection directly into the cerebrospinal fluid that bathes the spinal cord.

[0629] Treatment method Another aspect of the invention relates to a method of treatment comprising administering a viral vector of the invention or a cell transduced with a viral vector of the invention to a subject in need thereof.

[0630] All references herein to treatment include curative, palliative and prophylactic treatment, although it will be understood that in the context of the present invention, references to prevention more generally relate to prophylactic treatment. Treatment may also include the prevention or delay of disease progression. Mammalian treatment is particularly preferred. Both human and veterinary treatment are within the scope of the present invention.

[0631] In one embodiment, the viral vectors or viral vector particles of the invention may be for use as vaccines, which may be, for example, human or veterinary virus-based vaccines (e.g., influenza and Newcastle disease virus vaccines).

[0632] The present invention may be particularly useful when the vaccine is based on a modified competent virus carrying a transgene.

[0633] As mentioned above, avian producer cells can be used in the generation of viral vectors and viral vector particles for use as vaccines.

[0634] Pharmaceutical Compositions Another aspect of the present invention relates to a pharmaceutical composition comprising a viral vector of the present invention or a cell or tissue transduced with a viral vector of the present invention in combination with a pharmaceutically acceptable carrier, diluent or excipient.

[0635] The present invention provides a pharmaceutical composition for treating an individual by gene therapy, the pharmaceutical composition comprising a therapeutically effective amount of a vector. The pharmaceutical composition may be for human or veterinary use.

[0636] The composition can comprise pharmaceutically acceptable carriers, diluents, excipients or adjuvants.The selection of pharmaceutical carriers, excipients or diluents can be carried out according to the intended route of administration and standard pharmaceutical practice.The pharmaceutical composition can comprise any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilizing agent(s) and other carrier factors (such as lipid delivery systems) that can aid or increase the entry of vector into target site, or can be included in addition to carriers, diluents or excipients.

[0637] Where appropriate, composition can be administered by any one or more of inhalation; in the form of suppository or pessary; topically in the form of lotion, solution, cream, ointment or powder; by using skin patch; orally in the form of tablet containing excipients such as starch or lactose, or in capsule or vaginal suppository alone or mixed with excipients, or in the form of elixir, solution or suspension containing flavoring or coloring agent, or they can be parenterally injected, for example, intracavernosally, intravenously, intramuscularly, intracranially, intraocularly or subcutaneously.For parenteral administration, composition can be most suitably used in the form of sterile aqueous solution, which can contain other substances, for example, sufficient salt or monosaccharide to make the solution isotonic with blood.For buccal or sublingual administration, composition can be administered in the form of tablet or sweetened tablet, which can be formulated in a conventional manner.

[0638] The vectors of the present invention can also be used to transduce target cells or tissues ex vivo before introducing said target cells or tissues into a patient in need thereof. An example of such cells can be autologous T cells, and an example of such tissue can be a donor cornea.

[0639] Screening Methods Another aspect of the present invention relates to a method for identifying a nucleic acid binding site and / or a cognate nucleic acid binding protein that can interact such that when operably linked to the nucleic acid binding site, translation of a nucleotide of interest is suppressed or prevented in a viral vector producing cell, comprising analyzing expression of a reporter gene in a cell containing both the nucleic acid binding site and the nucleic acid binding protein operably linked to the reporter gene.

[0640] This method may allow for the identification of novel RNA-binding proteins and their corresponding binding sites that are useful in the present invention. This method may also allow for the identification of variants of known RNA-binding proteins or binding sites.

[0641] In one embodiment, the method allows for the identification of binding sites that interact with TRAP.

[0642] In another embodiment, the method allows for the identification of nucleic acid binding proteins that interact with a binding site capable of binding to TRAP.

[0643] In one embodiment, the reporter gene encodes a fluorescent protein.

[0644] In another embodiment, the reporter gene encodes a positive cell proliferation selection marker, such as the sh ble gene product, which confers cell resistance to Zeocin™.

[0645] In another embodiment, the reporter gene encodes a negative cell proliferation selection marker, such as the HSV thymidine kinase gene product, which causes cell death in the presence of gancyclovir.

[0646] An example of screening a TRAP binding site (tbs) for improved functionality can be as follows.

[0647] A degenerate DNA library containing 8 to 11 repeats of the sequence KAGNN or a total of 8 to 11 repeats of KAGNN and KAGNNN is synthesized.

[0648] The library is cloned within the 5' UTR (preferably within 12 nucleotides of the ORF) of a reporter gene cassette such as GFP. The reporter gene linked to the library can be optionally cloned into the retroviral vector genome and the resulting retroviral vector library.

[0649] The reporter gene cassette linked to the library is stably introduced into a cell line (this can be achieved by transfection or retroviral vector delivery) and single clones are isolated.

[0650] Screen clones by parallel transfection using control DNA (e.g., pBlueScript) or TRAP-expressing plasmid DNA. Measure reporter gene expression in both scenarios and identify clones with high unrepressed reporter gene levels (control) and low repressed reporter gene levels (TRAP).

[0651] Identify the tbs sequence by PCR amplification and sequencing of the tbs sequence from target cell genomic DNA from candidate clones.

[0652] The present invention will now be further described 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.

[0653] Polynucleotides The polynucleotide of the present invention can comprise DNA or RNA. The polynucleotide of the present invention can be single-stranded or double-stranded. It will be understood by those skilled in the art that due to the degeneracy of the genetic code, many different polynucleotides can encode the same polypeptide. Furthermore, it should be understood that those skilled in the art can use routine techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotide of the presen...

Claims

1. A nucleic acid comprising a nucleotide of interest and a tryptophan RNA-binding attenuation protein (TRAP) binding site, (a) the TRAP binding site overlaps the start codon ATG of the nucleotide of interest; and / or (b) the nucleic acid also contains a Kozak sequence, and the TRAP binding site overlaps with the Kozak sequence; the nucleotide of interest is operably linked to the TRAP binding site; Nucleic acid.

2. (a) the TRAP binding site is capable of interacting with a tryptophan RNA-binding attenuating protein such that translation of the nucleotide of interest is repressed in viral vector-producing cells; and / or (b) the nucleic acid of claim 1, wherein the nucleotide of interest is translated in a target cell lacking a tryptophan RNA-binding attenuating protein.

3. The TRAP binding site (a) Sequence KAGN ２－３ Contains multiple repetitions of; (b) Sequence KAGN ２ Contains multiple repetitions of; (c) Sequence KAGN ２ Contains at least six repeats of: (d) Sequence KAGN ２－３ comprising at least eight repeats of (e) Sequence KAGN ２－３ and the number of KAGNNN repeats is 1 or less; (f) Sequence KAGN ２ and / or comprising at least 8 to 11 repeats of (g) Sequence KAGN ２－３ and the number of KAGNNN repeats is 3 or less; The nucleic acid according to claim 1 or 2.

4. (a) the Kozak sequence and / or start codon overlaps the 3' end of the TRAP binding site; (b) the Kozak sequence and / or start codon overlaps with the 3'-terminal KAGNN repeat of the TRAP binding site; (c) the Kozak sequence comprises the sequence RNNATG (SEQ ID NO: 125) or RVVATG (SEQ ID NO: 28); (d) the overlapping Kozak sequence and / or start codon and TRAP binding site comprise any one of SEQ ID NOs: 29 to 33; (e) the nucleic acid comprises any one of SEQ ID NOs: 34-37, 69-92, 108-112, or SEQ ID NO: 114; and / or (f) the nucleic acid comprises any one of SEQ ID NOs: 34-37, 69-92, 108-112 or SEQ ID NO: 114, wherein the nucleic acid comprises one of SEQ ID NO: 34 or SEQ ID NO: 35; The nucleic acid according to any one of claims 1 to 3.

5. (a) The distance from the end of the transcription start site / promoter to the start of the TRAP binding site is 1 to 33 nucleotides long; (b) The distance from the end of the transcription start site / promoter to the start of the TRAP binding site is 1 to 12 nucleotides long: (c) the TRAP binding site lacks a type II restriction enzyme site; (d) the TRAP binding site lacks a SapI restriction enzyme site; (e) the nucleic acid comprises a 5' leader sequence upstream of the TRAP binding site; and / or (f) the nucleic acid comprises a 5' leader sequence upstream of the TRAP binding site; (i) the leader sequence comprises a sequence derived from the non-coding EF1α exon 1 region; or (ii) the leader sequence comprises the sequence defined in SEQ ID NO: 25 or SEQ ID NO: 26; The nucleic acid according to any one of claims 1 to 4.

6. The nucleic acid according to any one of claims 1 to 5, wherein the nucleic acid comprises an internal ribosome entry site (IRES).

7. The nucleic acid of claim 6 , wherein the nucleic acid comprises a spacer sequence between the internal ribosome entry site (IRES) and the TRAP binding site.

8. (a) the spacer sequence is 1 to 30 nucleotides in length; or (b) the spacer sequence is 15 nucleotides in length.

9. (a) the spacer comprises a sequence defined in any one of SEQ ID NOs: 38-44; and / or (b) the spacer comprises the sequence defined in SEQ ID NO:

39. The nucleic acid of claim 7 or 8.

10. The nucleic acid of any one of claims 1 to 9, wherein the nucleotide of interest produces a therapeutic effect.

11. 11. The nucleic acid of claim 1, wherein the nucleic acid further comprises an RRE sequence or a functional substitute thereof.

12. The nucleic acid according to any one of claims 1 to 11, wherein the nucleic acid is a vector transgene expression cassette.

13. (a) the 3'-terminal KAGNN repeat of the TRAP binding site overlaps with at least the first nucleotide of the ATG initiation codon; (b) the 3'-terminal KAGNN repeat of the TRAP binding site overlaps the first two nucleotides of the ATG start codon: (c) the 3'-terminal KAGNN repeat of the TRAP binding site overlaps the first nucleotide of the start codon ATG within the core Kozak sequence; and / or (d) the nucleic acid comprises the sequence defined in SEQ ID NO: 114 or SEQ ID NO:

116.

14. A viral vector comprising the nucleic acid of any one of claims 1 to 13.

15. The viral vector is (a) comprising more than one nucleotide of interest, wherein at least one nucleotide of interest is operably linked to a TRAP binding site as defined in any one of claims 1 to 3; (b) derived from a retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, or baculovirus; (c) derived from a lentivirus; and / or (d) derived from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or visna lentivirus; The viral vector of claim 14.

16. A viral vector production system comprising a set of nucleic acids encoding components required for the production of a viral vector, wherein the RNA genome of the viral vector comprises the nucleic acid of any one of claims 1 to 13.

17. (a) the viral vector is derived from a retrovirus, adenovirus, or adeno-associated virus; and / or (b) the viral vector is a retroviral vector; 17. The viral vector production system of claim 16, wherein the viral vector production system comprises nucleic acid sequences encoding Gag and Pol proteins, a tryptophan RNA-binding attenuation protein, and an Env protein, or functional substitutes thereof.

18. (a) the viral vector production system further comprises a nucleic acid sequence encoding rev or a functional substitute thereof; (b) the viral vector is derived from a lentivirus; and / or (c) the viral vector is derived from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or visna lentivirus; The viral vector production system of claim 17.

19. A DNA construct for use in the viral vector production system according to any one of claims 16 to 18, comprising the nucleic acid according to any one of claims 1 to 13.

20. A DNA construct for use in the viral vector production system of any one of claims 16 to 18, comprising a nucleic acid encoding a tryptophan-RNA binding attenuating protein.

21. A set of DNA constructs for use in the viral vector production system of any one of claims 16 to 18, comprising the DNA construct of claim 19, DNA constructs encoding Gag and Pol proteins, and a DNA construct encoding an Env protein or a functional substitute thereof.

22. 22. The set of DNA constructs of claim 21, wherein the set of DNA constructs further comprises a DNA construct encoding a rev sequence or a functional substitute thereof.

23. A viral vector-producing cell comprising the nucleic acid according to any one of claims 1 to 13, the viral vector production system according to any one of claims 16 to 18, the DNA construct according to claim 19, or a set of DNA constructs according to any one of claims 21 to 22.

24. (a) the cells are transiently transfected with a vector encoding a tryptophan-RNA binding attenuator protein; or (b) the cells stably express a tryptophan-RNA binding attenuator protein; The viral vector producing cell of claim 23.

25. A viral vector comprising the nucleic acid according to any one of claims 1 to 13, wherein the viral vector is produced by the viral vector production system according to any one of claims 16 to 18, or by using the viral vector-producing cell according to any one of claims 23 to 24.

26. (a) derived from a retrovirus, adenovirus, or adeno-associated virus; (b) derived from a lentivirus; and / or (c) the viral vector of claim 25, which is derived from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or visna lentivirus.

27. A cell transduced by the viral vector of claim 14, 15, 25 or 26.

28. A viral vector according to claim 14, 15, 25 or 26 or a cell according to claim 27 for use in medicine.

29. Use of a viral vector described in claim 14, 15, 25 or 26 or a cell described in claim 27 for the preparation of a medicament for delivering a nucleotide of interest to a target site requiring said nucleotide.

30. A pharmaceutical composition comprising the viral vector of claim 14, 15, 25 or 26 or the cell of claim 27 in combination with a pharmaceutically acceptable carrier, diluent or excipient.

31. A method for inhibiting translation of a nucleotide of interest (NOI) in a viral vector producing cell, the method comprising introducing into the viral vector producing cell a nucleic acid as defined in any one of claims 1 to 13 and a nucleic acid encoding a tryptophan-RNA binding attenuating protein (TRAP), wherein the TRAP binds to the TRAP binding site, thereby inhibiting translation of the NOI.

32. A method for increasing viral vector titer in a eukaryotic vector-producing cell, comprising introducing into the eukaryotic vector-producing cell a viral vector production system described in any one of claims 16 to 18 and a nucleic acid encoding a tryptophan-RNA-binding attenuating protein (TRAP), wherein the TRAP binds to the TRAP binding site and inhibits translation of the NOI, thereby increasing the viral vector titer compared to a viral vector not having a TRAP binding site.

33. The nucleic acid according to any one of claims 1 to 13, wherein the nucleic acid further comprises a promoter-5'UTR region.

34. (a) the TRAP binding site and the Kozak sequence, or the TRAP binding site, the multiple cloning site, and the Kozak sequence, are located within the 5'UTR of the promoter-5'UTR region; (b) the promoter-5'UTR region further comprises an intron; (c) the promoter-5'UTR region further comprises an intron, and the intron is upstream of the TRAP binding site; and / or (d) the promoter-5'UTR region is an engineered promoter containing a heterologous intron within the 5'UTR; 34. The nucleic acid of claim 33.

35. A nucleic acid encoding an RNA genome of a viral vector, wherein the RNA genome of the viral vector comprises a nucleic acid according to any one of claims 1 to 13, 33 or 34.

36. (a) a major splice donor site in the RNA genome of the viral vector is inactivated; (b) a major splice donor site and a cryptic splice donor site 3' to the major splice donor site in the RNA genome of the viral vector are inactivated; and / or (c) a major splice donor site and a cryptic splice donor site 3' to the major splice donor site in the RNA genome of the viral vector are inactivated, and the cryptic splice donor site is the first cryptic splice donor site 3' to the major splice donor site. A nucleic acid according to claim 35 or a viral vector production system according to claim 17 or 18.

37. (a) the cryptic splice donor site is within 6 nucleotides of the major splice donor site; and / or (b) the major splice donor site and the cryptic splice donor site are mutated or deleted; 37. A nucleic acid or viral vector production system according to claim 36.

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