Production system

Abstract

To provide a nucleic acid sequence that improves translation suppression of a nucleotide of interest (NOI) encoded by a viral vector.SOLUTION: The present invention provides a nucleic acid sequence comprising a nucleotide of interest and a tryptophan RNA-binding attenuation protein (TRAP) binding site, (i) wherein the TRAP binding site overlaps the ATG start codon of the nucleotide of interest; and / or (ii) wherein the nucleic acid sequence further comprises a Kozak sequence, and the TRAP binding site overlaps the Kozak sequence.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the production of viral vectors. More specifically, the present invention relates to the production of viral vectors. nucleotides of interest encoded by the viral vector in vector-producing cells. Regarding the modification of the translation of [Background technology]

[0002] Gene therapy broadly involves the use of genetic material to treat disease. A functional copy of a defective gene in a cell that has a defective gene (e.g., a gene with a mutation) These include replenishing genes, inactivating improperly functioning genes, and introducing new therapeutic genes. do.

[0003] Therapeutic genetic material is delivered to target cells of a host using vectors to allow the introduction of nucleic acids. Such vectors generally fall into the viral and non-viral categories. It can be divided into lees.

[0004] Viruses deliver their genetic material to target cells in the host as part of their replication cycle. The engineered viral vector naturally introduces the nucleotide of interest (NO) into the cell. I) or take advantage of this ability to enable the delivery of transgenes to target cells. To date, many viruses have been engineered as vectors for gene therapy. are retroviruses, adenoviruses (AdV), adeno-associated viruses (AAV), simplex viruses These include herpesvirus (HSV) and vaccinia virus.

[0005] In addition to being modified to carry an NOI, the viral vector is typically replication-deficient. Therefore, the recombinant vector cannot directly infect target cells. They can produce a number of viruses, but are unable to produce further generations of infectious virions. The virus vector may be conditionally replicable only in cancer cells and may contain a toxic transgene or promoter. It may further encode an enzyme.

[0006] Retroviral vectors are being developed as treatments for a variety of genetic disorders. Currently, clinical trials are showing increasing promise (e.g., Galy, A. and AJ .Thrasher(2010)Curr Opin Allergy Clin Im munol 11(6):545-550;Porter, DL, BLLevi ne, M.Kalos, A.Bagg and CHJune(2011)N En gl J Med 365(8):725-733;Campochiaro,PA (2012)Gene Ther 19(2):121-126;Cartier,N. ,S.Hacein-Bey-Abina,CC Bartholomae,P.Bo ugneres, M. Schmidt, CV Kalle, A. Fischer, M. Cavazzana-Calvo and P. Aubourg (2012)Metho ds Enzymol 507:187-198;Sadelain,M.,I.Riv iere, X. Wang, F. Boulad, S. Prockop, P. Giardin a, A. Maggio, R. Galanello, F. Locatelli and E. .Yannaki (2010) Ann NY Acad Sci 1202:52-5 8;DiGiusto,DL,A.Krishnan,L.Li,H.Li,SL i, A. Rao, S. Mi, P. Yam, S. Stinson, M. Kalos, JA. lvarnas,SFLacey,JKYee,M.Li,L.Couture ,D.Hsu,SJForman,JJRossi and JAZaia (2010)Sci Transl Med 2(36):36ra43 and Segu ra MM, MM, Gaillet B, Garnier A. (2013) Exp. ert opinion in biological therapy).

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

[0008] By reverse genetics, vectors encoding large heterologous sequences (approximately 10 kb) were prepared using suitable DNA fragments. These viruses were vectorized so that they could be produced by transfection of mammalian cells with the A sequence. It has become possible to significantly manipulate the vectors used as the base (Bannert, K. (20 10) Reviewed in Caister Academic Press: 347-370 (It is.)

[0009] The manipulation and use of retroviral vectors in research is typically performed using techniques such as G These clinical trials involve the production of reporter gene vectors encoding FP or lacZ. The titer of the unrelated vector is typically 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 transducing units (TU / mL). x10 10 Working stocks exceeding TU / mL can be achieved. Therefore, the production of vectors encoding therapeutically relevant NOIs can be achieved by using these reporter vectors. These often result in substantially reduced titers compared to

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

[0011] 1. Therapeutic genome size. Extremely large genomes can be packaged by retroviruses. Although reverse transcription and / or integration steps can be performed with increasing size, It is believed that this will result in lower efficiency.

[0012] 2. Stability of the vector genome RNA. This may be due to the presence of unexpected destabilizing elements in the NOI. This can be reduced by the presence of

[0013] 3. Suboptimal nucleotide usage in the vector genome RNA. The genome often has some nucleotide bias (e.g., HIV-1 is AT-rich). Vector genomes tend to be lower in AT richness, which may contribute to packaging and This may affect the maturation and / or post-maturation processes.

[0014] 4. Expression of NOI in viral vector-producing cells. (Over)expressed protein may have indirect or direct effects on vector virion assembly and / or infectivity. It is possible.

[0015] Expression of the protein encoded by the NOI in viral vector producing cells is Experience has shown that this can have a negative impact on the titer of therapeutic vectors (WO 2004 / 023166). (See issue 015 / 092440).

[0016] Vector for the protein encoded by the NOI (protein of interest, POI) Incorporation into virions can also affect downstream processing of the vector particle, e.g. The NOI encoding the transmembrane POI is inserted into the viral vector virion as a transmembrane protein. This could result in high surface expression of virions, potentially altering the physical properties of the virion. Incorporation of the POI may present the POI to the patient's immune system at the delivery site, which may have therapeutic effects in vivo. The NOI may be used for various purposes, including production, purification, Production of undesirable secondary proteins or metabolites that may affect recovery and immunogenicity It may also induce aging, so it is desirable to minimize this.

[0017] in viral vector producer cells while maintaining efficient expression of the NOI in target cells The ability to suppress the expression of the NOI of the virus is also desirable. The "natural" pathway of vector particle assembly and the resulting functionality must not be impeded. The viral vector genome molecules packaged into virions necessarily contain NOIs. This is not easy, as the current cassette must be coded. - The genomic molecule and the NOI expression cassette are operably linked, so that the NOI expression cassette Modifications of the vector may adversely affect the ability of the cell to produce vector genome molecules. For example, a physical transcription blocker (e.g., TetR) can be used to repress the NOI expression cassette. When a gene encoding a vector is used, the production of vector genome molecules is also inhibited due to steric hindrance. Furthermore, alterations in the regulatory mechanisms may also contribute to the development of vector virion maturation and release. - adversely affect the functionality of the genomic molecule (i.e., with respect to inducing transduction of target cells) For example, retroviral vector genomic RNA molecules must not be reverse transcribed and reassembled. The integration process must be possible and any modifications to the NOI expression cassette must be These steps in the introduction process must not be interrupted.

[0018] Suppression of NOI expression in viral vector-producing cells may offer additional advantages. If OI expression leads to a decrease in the viability of vector-producing cells, its suppression may be observed at large cell numbers. This may be beneficial for large-scale manufacturing, which requires a large amount of cell death. It also reduces impurities in purified vector harvest material. Standardize the processing, purification, and concentration of different therapeutic genes (encoded within the same vector system) It is possible that the only gene expressed in the viral vector-producing cells If the heterologous gene is a gene required for vector production, it is Downstream processing can be more easily optimized across platforms, and vector preparation The resulting vector in vivo will have very similar physical characteristics. This may minimize the variability of the immune response to and the toxicity of the resulting vector, which may result in increased production of target cells. This may result in more sustained therapeutic NOI expression in the cells.

[0019] Tissue-specific promoters that restrict expression of the NOI to the producing cells are a promising approach to this problem. Although a possible solution, the leakiness of these promoters can be detrimental. However, the use of a constitutive promoter can result in high levels of transgene protein. can be used to achieve greater and more robust expression of the NOI in the target cells Indeed, such robust expression may be required for efficacy in vivo. Tissue-specific promoters have been used in animal models and in human genomes during preclinical and clinical development. There may be less predictability when observing therapeutic vector production in vivo.

[0020] WO 2015 / 092440 (incorporated herein by reference) describes This suppresses translation of the NOI (repressing transgene expression) during virus vector production, thereby A method for inhibiting or preventing expression of a protein encoded by an NOI in eukaryotic cell culture This system discloses the use of a heterologous translational control system in vector production cell lines. Transgene suppression in T ransgene R Epression I n vector P The induction cell system (TRIP system) is one In its form, the TRIP system utilizes the bacterial trp operon to mediate transgene repression. Regulatory proteins, tryptophan RNA-binding decay protein (TRAP) and TRAP Surprisingly, the use of this system has does not interfere with the production of cloningable vector genome molecules or the activity of vector virions in target cells. Does not interfere with long-term expression of the NOI. [Prior art documents] [Patent documents]

[0021]

Patent Document 1

Non-licensed literature

[0022] [Non-licensed document 1] Galy,A.and AJThrasher(2010)Curr Opin Allergy Clin Immunol 11(6):545-550 [Non-licensed document 2] Porter,DL,BLLevine,M.Kalos,A.Bagg and CHJune(2011)N Engl J Med 365(8):725-733 [Non-licensed document 3] Campochiaro,PA(2012)Gene Ther 19(2):121-126

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

[0023] The present invention provides a method for improving the level of translational repression by TRAP, which can be used to improve the TRAP system. The present invention relates to modifications made to transgene mRNA that allow for improved transcription. The improved nucleic acid sequence may have, for example, the following characteristics:

[0024] 1. Consists of nucleotides derived from the first (non-coding) exon of the EF1α gene The improved 5'UTR leader sequence (upstream of tbs) surprisingly supports a variety of constructs. Compared with the 5'UTR leader sequence from the constitutive promoter, the TRAP-tbs complex allowing for consistently lower "repressed" levels of transgene expression mediated by It is shown that this is possible.

[0025] 2. An improved UT inserted between the internal ribosome entry site (IRES) and the tbs The R or "spacer" sequence surprisingly exhibited both fold suppression and non-suppression levels (i.e. It has been shown to improve both the effectiveness of the treatment (i.e., no TRAP) and the effectiveness of the treatment (i.e., no TRAP).

[0026] 3. The mutant Kozak sequence overlapping the 3' end of tbs surprisingly It is shown that this results in improved occlusion of the transgene start codon by the tbs complex.

[0027] 4. Pressure between tbs and the transgene Kozak sequence (transgene start codon (ATG)) The sequence containing the shortened, overlapping multiple cloning sites was surprisingly amenable to TRAP. facilitates cloning while retaining low levels of transgene expression when repressed by It is shown that this is possible.

[0028] 5. The overlap of the 3' end of tbs with the transgene initiation codon ATG is surprising. It was shown that the RAP-tbs complex results in improved occlusion of the transgene start codon. will be done.

[0029] In one aspect, the present invention provides a method for combining a nucleotide of interest with a tryptophan RNA binding enzyme. a nucleic acid sequence comprising a TRAP binding site and a Kozak sequence, The RAP binding site provides a nucleic acid sequence that 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. The Kozak sequence is a sequence encoding a tryptophan RNA-binding attenuating protein (TRAP) ) a nucleic acid sequence comprising a portion of the binding site.

[0031] In one aspect, the present invention provides a method for identifying a nucleotide sequence of interest (transgene) and a TRAP binding site. and a nucleic acid sequence comprising a TRAP binding site that includes a part of the transgene start codon ATG. or vice versa.

[0032] In some embodiments, the nucleotide of interest is a TRAP binding site or a portion thereof. The device is operatively connected to the

[0033] In some embodiments, translation of the nucleotide of interest is in a viral vector producing cell. The TRAP binding site, or a portion thereof, is inhibited in tryptophan RNA It can interact with binding attenuating proteins.

[0034] In some embodiments, the nucleotide of interest is tryptophan RNA binding attenuator. It is translated in target cells that lack the protein.

[0035] In some embodiments, the TRAP binding site or a portion thereof is Contains multiple repetitions.

[0036] In some embodiments, the TRAP binding site or portion thereof is selected from the group consisting of multiple sequences of the sequence KAGN2. This includes repetition of

[0037] In some embodiments, the TRAP binding site or portion thereof is at least one of the sequences of KAGN2. Contains at least six repeats.

[0038] In some embodiments, the TRAP binding site or a portion thereof is It contains at least 8 repeats. The number of KAGNNN repeats can be 1 or less.

[0039] In some embodiments, the TRAP binding site or portion thereof is at least one of the sequences of KAGN2. Contains at least 8 to 11 repeats.

[0040] In some embodiments, the TRAP binding site or a portion thereof is one of the sequences KAGN2-3. 1 repeat, and the number of KAGNNN repeats is 3 or less.

[0041] In some embodiments, the Kozak sequence is 3' of or a portion of the TRAP binding site. The Kozak sequence overlaps the 3' end of the TRAP binding site or part of it. May overlap with NN iterations.

[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 sites thereof Part of the 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 is SEQ ID NO: 69-92 or 108-112 The sequence shown in

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

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

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

[0051] In some embodiments, the nucleic acid sequence is located upstream of the TRAP binding site or a portion thereof. Contains a 5' leader 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. It may include.

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

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

[0054] In some embodiments, the spacer is defined by 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. nothing.

[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 a portion thereof in the TRAP binding site is The repeat overlaps at least the first nucleotide of the ATG start codon.

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

[0060] In some embodiments, the 3'-terminal KAGNN repeat of or a portion thereof in the TRAP binding site is The repeat sequence is the first nucleotide of the ATG start codon within the core Kozak sequence as defined herein. Overlaps with leotide.

[0061] In some embodiments, the nucleic acid sequence is defined in SEQ ID NO:114 or SEQ ID NO:116. It contains sequences that can be

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

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

[0064] In some embodiments, the viral vector is a retrovirus, an adenovirus, an adenovirus, or an adenovirus. Adeno-associated virus, herpes simplex virus, vaccinia virus, or baculovirus The viral vector may be derived from a lentivirus. HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or Visnare It may be derived from an antiviral agent.

[0065] In a further aspect, the present invention provides a method for producing a viral vector comprising the steps of: a viral vector production system comprising a set of nucleic acid sequences encoding the vector genome, A viral vector production system is provided, which comprises the nucleic acid sequence of the invention. It may be derived from a rovirus, an adenovirus or an adeno-associated virus.

[0066] In some embodiments, the viral vector is a retroviral vector. The vector production system contains Gag and Pol proteins, tryptophan RNA-binding attenuation factors, and Nucleic acid sequences encoding the Env protein, Env protein, or functional substitutes thereof Further includes:

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

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

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

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

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

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

[0073] In a further aspect, the present invention provides a method for producing a viral vector of the present invention by combining a nucleic acid sequence of the present invention with a viral vector of the present invention. A live system or a viral vector-producing cell comprising the DNA construct of the present invention is provided.

[0074] In some embodiments, the cells encode tryptophan-RNA binding decay proteins. The cells are transiently transfected with a vector encoding the tryptophan-RNA binding attenuation tag. The protein can be stably expressed.

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

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

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

[0078] In some embodiments, the viral vector is a retrovirus, adenovirus, or are derived from adeno-associated viruses. Viral vectors can be derived from lentiviruses. Viral vectors include HIV-1, HIV-2, SIV, FIV, BIV, EIAV, C It can be derived from AEV or visna lentivirus.

[0079] In a further aspect, the present invention provides a method for producing a virus transduced by a viral vector of the present invention. Provide the cells.

[0080] In a further aspect, the present invention provides a viral vector of the invention for use in medicine. The present invention provides a method for producing a medicament for the treatment of a cancer, comprising the steps of:

[0081] In a further aspect, the present invention provides a method for identifying a target site requiring a nucleotide of interest. The viral vectors or vectors of the present invention for the preparation of a medicament for delivering a nucleotide to a target or uses of the cells of the invention.

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

[0083] In a further aspect, the present invention provides a method for administering the viral vector of the present invention or the cell of the present invention to a patient. and a pharmaceutical composition comprising the compound of formula (I) in combination with a physiologically acceptable carrier, diluent or excipient. do.

[0084] In a further aspect, the present invention provides a method for the preparation of a nucleic acid-binding domain of interest, comprising the steps of: The translation of the nucleotides of the vectors is inhibited in viral vector-producing cells. A method for identifying nucleic acid binding sites and / or nucleic acid binding proteins capable of , both a nucleic acid binding site and a nucleic acid binding protein operably linked to a reporter gene and analyzing expression of a reporter gene in a cell comprising the gene.

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

[0086] In a further aspect, the present invention provides a method for producing a nucleic acid of interest in a viral vector producing cell. A method for inhibiting translation of a NOI comprising the steps of: The nucleic acid sequence encoding the Fan-RNA-binding attenuation protein (TRAP) was inserted into a viral vector. - including introducing into the producing cell, TRAP binding to the TRAP binding site or a part thereof and thereby inhibiting translation of the NOI.

[0087] In a further aspect, the present invention provides a method for producing a viral vector in a eukaryotic vector-producing cell. A method for increasing the titer of a virus of the present invention in a eukaryotic vector-producing cell, comprising: Vector production system and tryptophan-RNA binding decay protein (TRAP) encoding and introducing a nucleic acid sequence that binds to the TRAP binding site or a portion thereof. and inhibits translation of the NOI, thereby preventing the viral vector from having a TRAP binding site. The present invention provides a method for increasing viral vector titer compared to a conventional method.

[0088] In a further aspect, the invention provides a method for producing a tryptophan-RNA fragment comprising the steps of: A binding site for TRAP, a multiple cloning site, and a Kozac a nucleic acid sequence comprising a multiple cloning site and a sequence, the multiple cloning site being located below the TRAP binding site; The nucleic acid sequence is provided, which is located upstream of the stream and Kozak sequences.

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

[0090] In some embodiments, the promoter further comprises an intron, preferably an intron. The promoter is located upstream of the TRAP binding site or part of it. It may be an engineered promoter containing a heterologous intron.

[0091] In some embodiments, the nucleic acid sequence of the invention is SEQ ID NO: 117, 118 or 120. to 124.

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

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

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

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

[0096] In some embodiments, the major splice donor in the RNA genome of the viral vector The nucleotide sequence of the major splice donor site and the cryptic splice donor site 3' to the major splice donor site are inactive. It's sexualized.

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

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

[0099] In some embodiments, the major splice donor site and the cryptic splice donor site The position is mutated or deleted.

[0100] In some embodiments, the RNA genomic DNA of the viral vector prior to inactivation of the splice sites is Nucleotide sequences encoding the genoms are SEQ ID NOs: 94, 96, 97, 102, 103 and and / or 106.

[0101] In some embodiments, the nucleotides encoding the RNA genome of the viral vector The sequence may be any of SEQ ID NOs: 94, 96, 97, 102, 103 and / or 106. Includes sequences that have mutations or deletions relative to the sequence shown.

[0102] In some embodiments, the nucleotides encoding the RNA genome of the viral vector The sequence corresponds to nucleotides 13 and 14 of SEQ ID NO:94 if not inactivated. It contains an inactivated major splice donor site with a cleavage site between the nucleotides.

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

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

[0105] In some embodiments, the nucleotides encoding the RNA genome of the viral vector The sequence corresponds to nucleotides 17 and 18 of SEQ ID NO:94 if not inactivated. It contains an inactivated cryptic splice donor site with a cleavage site between the nucleotides.

[0106] In some embodiments, the nucleotides encoding the RNA genome of the viral vector The sequences are SEQ ID NOs: 95, 98, 99, 100, 101, 104, 105 and / or 1 07.

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

[0108] In some embodiments, the major splice donor portion of the RNA genome of the viral vector. Splicing activity from the nucleotide sequence and cryptic splice donor sites is suppressed or eliminated. .

[0109] In some embodiments, the major splice donor portion of the RNA genome of the viral vector. Splicing activity from the nucleotide sequence and cryptic splice donor sites was measured in transfected cells. or in 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 be understood to encompass, unless otherwise indicated, chemical, molecular biology, and other techniques within the capabilities of those skilled in the art. For the detection of HIV-1, conventional techniques of microbiology and immunology are used. Such techniques are described in the literature. For example, J. Sambrook, E.F. Fritsch, and T. Mania tis(1989)Molecular Cloning:A Laboratory Manual,Second Edition,Books 1-3,Cold Spr ing Harbor Laboratory Press;Ausubel,FM et al.(1995 and periodic supplements)Cur rent Protocols in Molecular Biology,Ch.9 ,13,and 16,John Wiley&Sons,New York,NY;B .Roe, J. Crabtree, and A. Kahn (1996) DNA Isol ation and Sequencing:Essential Technique s,John Wiley&Sons;JMPolak and James O' D. McGee (1990) In Situ Hybridization: Princ. iples and Practice;Oxford University Pre ss;MJGait(ed.)(1984)Oligonucleotide Sy nthesis:A Practical Approach,IRL Press;a nd,DMJLilley and JEDahlberg(1992)Me thods of Enzymology:DNA Structure Part A :Synthesis and Physical Analysis of DNA See Methods in Enzymology, Academic Press. 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, but rather by the Any methods and materials similar or equivalent to those described herein may be used to practice or otherwise adapt the disclosed embodiments. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, each nucleic acid sequence is written left to right in the 5' to 3' direction. , amino acid sequences are written left to right in the amino to carboxy direction.

[0115] Where a range of values ​​is given, any value between the upper and lower limits of that range, unless the context clearly indicates otherwise, Unless otherwise specified, each intervening value to the nearest tenth of a unit shall also be specifically disclosed. Any stated value or intervening value within a stated range and the Each smaller range between any other stated value or intervening value within a range is within the scope of the present disclosure. The upper and lower limits of these smaller ranges may independently be included within the range. Both may be included or excluded, with the smaller range including either limit. Each range that does not include one limit or that includes both limits also includes any particular The ranges set forth are included within the present disclosure as though they were within the limits specifically excluded. If one or both of the limits are included, a range excluding one or both of the included limits is also included in this disclosure. Included in the display.

[0116] As used in this specification and the appended claims, the singular forms "a," "an," "an" and "an" are used interchangeably. " and "the" include plural referents unless the context clearly dictates otherwise. Please note:

[0117] As used herein, "comprising" and "comprise" The terms "comprised of" and "comprised of" are used interchangeably with "including" and "comprised of." "including," "includes," or "contains" Synonymous with "containing" or "contains" and is inclusive or non-exclusive The term is inclusive and does not exclude additional, unrecited members, elements or method steps. "comprising", "comprises" and "consists of" "comprised of" is a reference to the term "consisting of" f) is also included.

[0118] The publications discussed herein are incorporated by reference in their entirety for their disclosure prior to the filing date of the present application. Any mention herein of such publication is provided solely as a guide to the publication to which it pertains. should not be construed as an admission that any of the foregoing constitutes prior art against the claims. do not have.

[0119] Nucleic acid sequence In one aspect, the present invention provides a method for combining a nucleotide of interest with a tryptophan RNA binding enzyme. a nucleic acid sequence comprising a TRAP binding site and a Kozak sequence, The RAP binding site (tbs) provides a nucleic acid sequence that overlaps with the Kozak sequence.

[0120] In one aspect, the present invention provides a method for producing a gene encoding a nucleotide of interest (transgene) and a tryptophan-containing gene. a nucleic acid sequence comprising a transcription factor-binding protein (TRAP) binding site (tbs); The TRAP binding site provides a nucleic acid sequence that 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. The Kozak sequence is a sequence that binds to tryptophan RNA-binding decay protein (TRAP). A nucleic acid sequence containing a portion of the binding site (tbs) is provided.

[0122] In one aspect, the present invention provides a method for identifying a nucleotide sequence of interest (transgene) and a TRAP binding site. and a nucleic acid sequence comprising the TRAP binding site (tbs) and the transgene start codon AT Nucleic acid sequences containing a portion of G, or vice versa, are provided.

[0123] In some embodiments of the present invention, the nucleotide of interest is tbs or a portion thereof. In some embodiments, the nucleotide of interest is operably linked to TR It is translated in target cells that lack AP.

[0124] tbs or a portion thereof, wherein translation of the nucleotide of interest is It may be possible that the protein interacts with TRAP such that it is inhibited or prevented in the presence of TRAP.

[0125] Therefore, in another aspect, the present invention provides a method for producing a viral vector by expressing an NOI in a viral vector producing cell. A method for inhibiting translation of a nucleic acid sequence of the present invention, comprising: The TRAP is introduced into a viral vector-producing cell, and the TRAP is introduced into a TRAP binding site or The present invention provides a method for inhibiting translation of an NOI by binding to a target gene or a portion thereof.

[0126] Tryptophan RNA-binding decay protein (TRAP) Tryptophan RNA-binding decay protein (TRAP) is a tryptophan RNA-binding decay protein (TRAP) expressed in Bacillus subtilis (B It is a bacterial protein that has been extensively characterized in Acillus subtilis. Tryptophan RNA-binding attenuation proteins (TRAPs) are involved in transcriptional attenuation or translational regulation. The trpEDCFBA operon directs the transcription of the ribosomal endothelial cells by inducing transcription of the ribosomal endothelial cells. Regulating tryptophan biosynthesis (Gollnick, B., Antson, and Y anofsky(2005)Annual Review of Genetics 3 9:47-68).

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

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

[0129] 2. Promotion of the formation of the trpE and trpD Shine-Dalgarno-blocking hairpins (Yak hnin H, B.J., Yakhnin AV, Babitzke P. (2001). Journal of Bacteriology 183(20):5918-592 6).

[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 177:4272-42 78).

[0131] In Bacillus subtilis, TRAP is a single The functional protein is encoded by the gene (mtrB) and arranged as a toroidal ring. It is composed of 11 identical subunits (Antson AA, DE, D odson G, Greaves RB, Chen X, Gollnick P. (19 99) Nature 401(6750):235-242). TRAP is activated , 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. (19 94) Journal of Biological Chemistry 269:1 6597-16604).

[0132] Without wishing to be bound by theory, it is believed that tryptophan synthesis is sensed and controlled by the In the natural mechanism, TRAP binds to a binding site in the newly synthesized RNA leader. It is understood that it acts at the level of transcription termination by binding to downstream rho non- Destabilizes the overlapping anti-terminator sequence so that the dependent terminator is active. , resulting in the production of only short RNAs. When tryptophan is limiting in bacteria, Once this occurs, the TRAP ring can no longer bind to its RNA binding site. The transinator is activated and transcription continues to the tryptophan synthesis gene operon. It can also act at the translational level: binding of TRAP in the 5'-UTR of RNA transcripts. Tryptophan-dependent binding of TRAP to the binding site releases the anti-Shine-Dalgarno sequence. This forms a stable stem with the Shine-Dalgarno sequence, resulting in its binding to the ribosome. Finally, other situations in which TRAP is bound to its tbs In , the 40S scanning ribosome complex can reach the start codon, The 40S strand is formed immediately before the otherwise more stable, higher affinity translation machinery. It is possible to inhibit translation initiation by physically blocking the Canning ribosome complex. can.

[0133] Because bacterial gene sequences are likely not optimal for expression in mammalian cells, TRAP Open reading frames are found in mammals (e.g., Homo sapiens). The sequence may be codon-optimized for expression in piens cells. The TRAP transcript can also be optimized by removing unnecessary sequences and splice sites. The use of a C-terminally expressed HIS tag on a protein provides benefits for translational repression. This C-terminal HIS tag appears to facilitate the dissolution of TRAP in eukaryotic cells and may be used. Although it may improve the biodegradability or stability, it cannot eliminate the improved functional benefits. Nevertheless, both HIS-tagged and untagged TRAPs were able to induce transgene expression. Certain cis-acting sequences within the TRAP transcription unit also allow for robust repression of the gene. For example, constructs driven by the EF1α promoter can be used for transient expression. In the transfection situation, TRA was significantly reduced compared to the CMV promoter-driven construct. This allows for better suppression at lower inputs of the P plasmid.

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

[0135] In one embodiment of the invention, the TRAP is a Bacillus species, e.g., a bacterium. It is derived from Bacillus subtilis. For example, TRA P may comprise the following sequence:

[0136] MNQKHSSDFVVIKAVEDGVNVIGLTRGTDTKFHHSEKLD KGEVIIAQFTEHTSAIKVRGEALIQTAYGEMKSEKK(Sequence number No. 1) In a preferred embodiment of the invention, SEQ ID NO: 1 is C-terminally terminated with six histidine amino acids. Tagged (HIS x 6 tags).

[0137] In an alternative embodiment, TRAP is produced by Aminomonas paucivorans (Aminomonas For example, a TRAP may include the following sequence: do.

[0138] MKEGEEAKTSVLSDYVVVKALENGVTVIGLTRGQETKFA HTEKLDDGEVWIAQFTEHTSAIKVRGASEIHTKHGMLFSG RGRNEKG (SEQ ID NO: 2) In an alternative embodiment, TRAP is Desulfotomaculum hydrothermalum (Desu lfotomaculum hydrothermale). For example, TRA P may comprise the following sequence:

[0139] MNPMTDRSDITGDYVVVKALENGVTIIGLTRGGVTKFHH TEKLDKGEIMIAQFTEHTSAIKIRGRAELLTKHGKIRTEV DS (SEQ ID NO: 3) In an alternative embodiment, the TRAP is derived from Bacillus stearothermophilus (B. stearothermophilus). For example, a TRAP may include the following sequence: do.

[0140] MYTNSDFVVIKALEDGVNVIGLTRGADTRFHHSEKLDKG EVLIAQFTEHTSAIKVRGKAYIQTRHGVIESEGKK(SEQ ID NO: 4) In an alternative embodiment, TRAP is produced by Bacillus stearothermophilus S72N (B. stearothermophilus S72N). For example, TRAP is It may include the following sequences:

[0141] MYTNSDFVVIKALEDGVNVIGLTRGADTRFHHSEKLDKG EVLIAQFTEHTSAIKVRGKAYIQTRHGVIENEGKK(SEQ ID NO: 5) In an alternative embodiment, TRAP is produced by Bacillus halodurans (B. halodurans For example, a TRAP may comprise the following sequence:

[0142] MNVGDNSNFFVIKAKENGVNVFGMTRGTDTRFHHSEKLD KGEVMIAQFTEHTSAVKIRGKAIIQTSYGTLDTEKDE(sequence Number 6) In an alternative embodiment, TRAP is derived from Carboxydothermus hydrogenoformans. (Carboxydothermus hydrogenoformans) For example, the TRAP may comprise the following sequence:

[0143] MVCDNFAFSSAINAEYIVVKALENGVTIMGLTRGKDTKF HHTEKLDKGEVMVAQFTEHTSAIKIRGKAEIYTKHGVIKN E (SEQ ID NO: 7) In one embodiment, TRAP is a member of the tryptophan RNA-binding attenuating protein gene family. mtrB (e.g., domain database #cl03437 stored in NCBI) It is encoded by the TrpBP superfamily.

[0144] In a preferred embodiment, the TRAP is tagged at the C-terminus with six histidine amino acids. (HIS x 6 tags).

[0145] In a preferred embodiment, TRAP is a nucleotide sequence selected from the group consisting of 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270 0%, 80%, 90%, 95%, 99% or 100% identity and operably linked The expression of the NOI may be modified in the viral vector producing cells, e.g., suppressed or The RNA-binding site comprises an amino acid sequence capable of interacting with the RNA-binding site so as to inhibit or prevent the binding of the RNA.

[0146] In a preferred embodiment, the TRAP has at least 50% identity with any of SEQ ID NOs: 1 to 7. 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, Expression of operably linked NOIs with 99% or 100% identity is achieved by viral vectors. RNA binding is altered, e.g., inhibited or prevented, in target-producing cells. It comprises an amino acid sequence capable of interacting with the site.

[0147] In another embodiment, the TRAP is a vector in which expression of the operably linked NOI is mediated by a viral vector. The RNA binding moiety may be modified, e.g., suppressed or prevented, in the producing cell. A polynucleotide comprising a nucleotide sequence encoding a protein capable of interacting with the site. For example, TRAP can encode the proteins of SEQ ID NOs: 1 to 7. It can be encoded by a polynucleotide comprising the encoding nucleotide sequence.

[0148] Any variant, fragment or homologue of TRAP for use in the present invention may be Translation of I (which may be a marker gene) is suppressed or The nucleotides retain the ability to bind to the TRAP binding site described herein so as to be prevented.

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

[0150] A consensus TRAP binding site sequence that can bind TRAP may bind multiple times (e.g., Repeated (e.g., 6, 7, 8, 9, 10, 11, 12 or more times) N], and such sequences are found in the natural trp operon. Occasionally, AAGNN is tolerated, and occasionally additional "spacing" N nucleotides are functional. In vitro experiments have demonstrated that TRAP-RNA binding is at least 6 or 7 times more efficient than the nucleotide sequence. demonstrate that many more consensus repeats are required (Babitzke P, Y. J., Campanelli D. (1996) Journal of Ba. cteriology 178(17):5159-5163). Therefore, it is preferable In one embodiment, 6 or more consecutive [KAGN ≧2 ]array 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 TRIP system preferably comprises at least It also works maximally with tbs sequences containing eight KAGNN repeats, but using seven repeats and still obtain robust transgene silencing. Using six repeats, The transgene can be sufficiently suppressed to a level that the target titer can be rescued. The KAGNN consensus sequence may be altered to maintain TRAP-mediated repression, Preferably, the exact sequence selected ensures high levels of translation in the unrepressed state. For example, the tbs sequence may be optimized to splice sites, unstable sequences or splices that can interfere with the translation efficiency of mRNA (in cells) The structure of the KAGNN iterations for a given tbs can be optimized by removing the loops. For the construction, the number of N "spacing" nucleotides between KAG repeats is preferably 2. However, there are more than two N spacers between at least two KAG repeats. tbs with 3 N as judged by in vitro binding studies are acceptable As many as 50% of repeats containing tbs can result in functional tbs; Babitzke P, YJ ., Campanelli D. (1996) Journal of Bacterio logy 178(17):5159-5163). In fact, the 11×KAGNNtbs array allows up to three substitutions with KAGNNN repeats and how many are combined with TRAP binding. It has been shown that some potentially useful translation blocking activity can still be retained.

[0152] In one embodiment of the invention, the TRAP binding site or a portion thereof has the sequence KAGN ≧2 (example For example, KAGN 2~3 ) Therefore, for the avoidance of doubt, this TBS or its Part of the repeat sequence UAGNN, GAGNN, TAGNN, UAGNN N, 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 Preferably 2, but up to 3, e.g. 1, 2 or 3, 11 x repeat tbs or Some KAG repeats may be separated by three spacing nucleotides, Preferably, the protein may still retain some TRAP binding activity that results in translational repression. To maintain maximum TRAP binding activity, 11 repeat tbs or a portion thereof No more than one N3 spacer will be used in the

[0154] In another embodiment, the tbs or a portion thereof is ≧2 Multiple iterations of (e.g., K AGN 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 repetitions of ( For example, KAGN 2~3 (at least six repeats of

[0157] In another embodiment, the tbs or portion thereof comprises at least six repeats of KAGN2. For example, tbs or parts thereof are KAGN2 6, 7, 8, 9, 10, 11, 12 For example, tbs or a portion thereof may contain SEQ ID NO: 8 to 10 or more repeats. It may include any one of 19 or 22.

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

[0159] Preferably, the number of KAGNNN repeats present in a tbs or part thereof is 1 or For example, tbs or a portion thereof may be selected from the group consisting of SEQ ID NOs: 8, 9, 14-17, 19 may include any one of 1 to 24.

[0160] In another embodiment, the tbs or a portion thereof is ≧2 11 iterations of (e.g., K AGN 2~3 Preferably, the tbs or a portion thereof contains The number of KAGNNN iterations to be used is 3 or less. For example, tbs or a part of it. may include 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., K AGN 2~3 (12 repetitions of the

[0162] In a preferred embodiment, the tbs or a portion thereof is selected from the 8-11 repeats of KAGN2 (e.g., For example, 8, 9, 10 or 11 repeats of KAGN2. The portion may include any one of SEQ ID NOs: 8, 9, 14-17, and 20-24.

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

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

[0165] GAGUUUAGCGGAGUGGAGAAGAGCGGAGCCGAGCCUAGC AGAGACGAGUGGAGCU (SEQ ID NO: 8); or GAGUUUAGCGGAGUGGAGAAGAGCGGAGCCGAGCCUAGC AGAGACGAGAAGAGCU (SEQ ID NO: 9) "KAGN ≧2 "Iteration" is a general KAGN ≧2 (e.g., KAGN 2~3 ) Mochi It should be understood that the motif is repeated. Different KAGNs that meet the criteria of this motif ≧ The two sequences may be linked to form a tbs or a portion thereof. It is not intended that the subset be limited to a single repeat of a sequence that meets the requirements of this motif. Although not specified, this possibility is included in the definition. ≧2 Six iterations of includes, but is not limited to, the following sequences: UAGUU-UAGUU-UAGUU-UAGUU-UAGUU-UAGUU(sequence number No. 10); UAGUU-UAGUU-GAGUU-UAGUU-GAGUU-UAGUU(Sequence number No. 11); GAGUUU-GAGUU-GAGUU-GAGUUU-GAGUU-GAGUU (arrangement Column number 12) and UAGUUU-GAGUU-UAGUU-GAGUUU-UAGUU-GAGUU(Arrangement Column number 13) (The dashes are included here between the repeats for clarity only).

[0166] One KAGNNN repeat and one 8-repeat tbs containing seven KAGNNN repeats or one of The portion retains TRAP-mediated repression activity. One or more KAGNNN repeats containing less than 8 repeat tbs sequences or portions thereof (e.g., 7 or 6 repeat tbs sequences or 8 or a portion thereof may have lower TRAP-mediated inhibitory activity. If there are no repeats present, it is preferred that the tbs or part of it contains only KAGNN repeats. It's nice.

[0167] Preferred nucleotides for use in the KAGNN repeat consensus are: This is the case.

[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 G is used in the K position when the NN spacer position is AA ( That is, TAGAA is preferably not used as a repeat in the consensus sequence. ).

[0170] "Able to interact" means that the nucleic acid binding site (e.g., tbs or a portion thereof) However, under conditions encountered in cells, e.g., eukaryotic viral vector producing cells, the protein It should be understood that the protein can bind to a protein such as TRAP. Such interactions with any RNA-binding protein involve nucleic acid binding sites (e.g., tbs or This results in the repression or prevention of translation of the NOI to which it is operably linked.

[0171] "Operably linked" means that the described components are linked together in their intended manner to form the described components. Therefore, N The tbs or portion thereof for use in the present invention operably linked to OI may be a TR The NOI is positioned so that translation is modified when AP binds to the tbs or part of it. are.

[0172] A TR is located upstream of the NOI translation initiation codon of a given open reading frame (ORF). By placing the tbs or parts of it that can interact with the AP, This allows for specific translational repression of mRNA derived from RF. The number of nucleotides separating the start codons can be varied without affecting the degree of suppression, e.g., 0 It can vary from 1 to 34 nucleotides. From 0 to 13 nucleotides may be used to separate a portion of the sequence from the translation initiation codon.

[0173] tbs or parts thereof repress the translation of NOIs in multicistronic mRNAs For this purpose, it can be placed downstream of an internal ribosome entry site (IRES). Further evidence that TRAP bound to tbs can block passage of the 40S ribosome is provided. IRES elements provide a CAP-independent pathway for transcription prior to the formation of the complete translation complex. It functions to enclose the 40S ribosomal subunit in mRNA in an IRES manner (IRES translation). For a review of translation initiation, see Thompson, S. (2012) Trends in (See Microbiology 20(11):558-566). Therefore, The TRIP system allows the synthesis of multiple autologous vectors from a single mRNA expressed from a viral vector genome. It is possible to suppress multiple reading frames. When preparing vectors for transfection, it is important to ensure that all transgene products are in the titer of the vector. This is a useful feature of the TRIP system when the effects of steroids on the immune system can be adversely affected to some degree.

[0174] In one embodiment, the nucleic acid sequence has a spacer sequence between the IRES and the tbs or a portion thereof. IRES is referred to herein under the subheading "Internal Ribosome Entry Site." The spacer sequence may be an IRES as described above. The spacer sequence may be 0 to 30 nucleotides in length, preferably The spacer may be 15 nucleotides in length. The spacer may be defined as any one of SEQ ID NOs: 38 to 44. Preferably, the spacer comprises the sequence defined in SEQ ID NO: 38. nothing.

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

[0176] In one embodiment, the tbs or portion thereof lacks a type II restriction enzyme site. In embodiments, 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. nothing.

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

[0179] Overlapping Kozak sequences and TRAP binding sites We found, surprisingly, that compared to the use of non-overlapping tbs and Kozak sequences (using overlapping tbs and Kozak sequences; see Figure 2B and Figure 2C) Improved inhibition can be achieved by "hiding" a Kozak sequence within the 3' end of s or a portion thereof. Furthermore, unexpectedly, we found that the overlapping Kozak and tbs sequences induced efficient levels of translation initiation, i.e., all of the sequences tested The overlapping sequences induce similar levels of induction as the non-overlapping Kozak and tbs sequences in the absence of TRAP. Without wishing to be bound by theory, improved suppression of transgene expression was observed. The observed levels are the TRAP-tbs complexes when the tbs or part of it overlaps with the Kozak sequence. This may be due to improved occlusion of the transgene start codon by the conjugation.

[0180] The term "Kozak sequence" refers to the sequence recognized by the ribosome as the translation initiation site. It should be understood as a consensus sequence in nuclear mRNA. Contains an ATG initiation (start) codon in NA (AUG in mRNA). The precise Kozak sequence present in A determines the efficiency of translation initiation, i.e., certain Kozak sequences The block sequence does not result in efficient translation initiation.

[0181] The complete Kozak sequence is typically the consensus sequence (gcc) for DNA. ccRccATGG and for RNA the consensus sequence (gcc) gccRccA UGG, where: the lower case indicates the maximum number of bases at this position that can be changed. represents a common base, an uppercase letter represents a highly conserved base at this position, and "R" indicates that a purine (i.e., A or G) is typically optimal at this position, The sequence within the arc (gcc) is of uncertain importance. T / U is generally located upstream of the start codon. It is the least preferred nucleotide at every position in a given Kozak sequence consensus.

[0182] The significance of the first three bases of the complete Kozak sequence is uncertain, so the Kozak sequence is , a consensus sequence referred to herein as the "extended Kozak sequence," i.e., DN GNNRVVATGG (SEQ ID NO: 27) for A and GNNRV for RNA VAUGG, and "R" indicates a purine (i.e., It should be understood that "V" designates any of the following: G, A, or C. It should be understood to designate any nucleotide, and "N" may be any nucleotide at its position in the sequence. For example, "N" refers to G, A, T, It can be C or U. The "R" at position -1 and the "G" at position +3 (the "A" in ATG) It was noted that the position 0 is considered the most important position in terms of Kozak strength. However, the presence of a "G" at position +3 in the transgene sequence Depending on the ORF, the +3 position is considered to be part of the "core" Kozak sequence in this specification. It is not considered as such.

[0183] The bases found in the first six positions of the complete Kozak sequence are those in which any base may be present. The change is made so that the group can be found (noted above as (gcc)gcc). Therefore, The complete Kozak consensus sequence is the reduced variability sequence denoted RccAUG above. It is believed that the Kozak sequence contains a "core" Kozak sequence consisting of a portion of the complete Kozak sequence having The "core" Kozak consensus sequence is used herein as R for mRNA. VVAUG and RVVATG for DNA, where "R" is the It should be understood that "V" designates a purine (i.e., A or G) at that position. " should be understood to designate any nucleotide from G, A, or C.

[0184] In one preferred embodiment of the present invention, the Kozak sequence is the sequence RVVATG (SEQ ID NO: 28), where "R" represents 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. It should be understood to specify

[0185] In one embodiment of the invention, the Kozak sequence comprises the sequence RNNATG (SEQ ID NO: 125). where "R" designates a purine (i.e., A or G) at that position in the sequence. It should be understood that "N" refers to any nucleotide from G, A, T / U or C. It should be understood that the use of "T / U" indicates low expression in the absence of TRAP. It is recognized that this may result in lowered levels.

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

[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 contains at least the first or first two ATG triplets in the core Kozak sequence. Overlapping nucleotides.

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

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

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

[0192] In one aspect, the overlapping tbs-Kozak sequences are GAGA, as shown in FIG. 2C. TG (SEQ ID NO: 29).

[0193] In one embodiment, the 3'-terminal KAGNN repeat of or part of the TRAP binding site is The target nucleotide overlaps with the first nucleotide of the ATG triplet.

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

[0195] In one embodiment, overlapping Kozak sequences and T The RAP binding site or a portion thereof may have 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 and "R" represents a purine at that position in the sequence (i.e. , A, or G), and "V" should be understood to designate any of G, A, or C. "N" should be understood to designate any nucleotide at that position in the sequence. For example, "N" should be understood to designate any of the nucleotides G, A, T, C, and , 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 and "R" represents a purine at that position in the sequence (i.e. , A, or G), and "V" should be understood to designate any of G, A, or C. "N" should be understood to designate any nucleotide at that position in the sequence. For example, "N" should be understood to designate any of the nucleotides G, A, T, C, and , or U.

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

[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: where "K" can be T or G and "N" can be any nucleotide at that position in the sequence. This should be understood to specify the code. For example, this could be G, A, T, C, or U. It is possible.

[0200] Preferably, the nucleic acid sequence is the following sequence: (a) KAGCCGAGATG (SEQ ID NO: 34); or (b) KAGNGGAGCCATG (SEQ ID NO: 35); including one of the following: where "K" can be T or G and "N" can be any nucleotide at that position in the sequence. This should be understood to specify the code. For example, this could be G, A, T, C, or U. It is possible.

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

[0202] The inhibition or prevention of translation of the NOI occurs at a comparable time in the absence of the nucleic acid sequence of the present invention. The amount of NOI product (e.g., TAGA) translated during viral vector production compared to the amount expressed Such a change in translation should be understood as a change in the amount of NOI. This results in the inhibition or prevention of expression of the encoded protein.

[0203] In one embodiment, the nucleic acid sequences of the present invention are designed such that translation of the nucleotide of interest is carried out in a viral vector. It can interact with TRAP in a manner that is inhibited or prevented in ATP-producing cells. do.

[0204] The translation of the NOI at any given time during vector construction is 90%, 80%, 70%, 60%, or 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or can be reduced to 0.1%.

[0205] The translation of the NOI at any given time during vector construction is 90%, 80%, 70%, 60%, or 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or can be reduced to less than 0.1%.

[0206] In the context of the present invention, translation of the NOI at any given time during vector construction is At the same time during target construction, non-overlapping Kozak and (unlike the nucleic acid sequences of the present invention) and 90%, 80%, 70%, or 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0 It can be reduced to 0.5% or 0.1%.

[0207] In the context of the present invention, translation of the NOI at any given time during vector construction is At the same time during target construction, non-overlapping Kozak and (unlike the nucleic acid sequences of the present invention) and 90%, 80%, 70%, or 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0 It can be reduced to less than 0.5% or 0.1%.

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

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

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

[0211] In the context of the present invention, the protein from the NOI at any given time during vector construction Expression of the target gene is overlapped (unlike the nucleic acid sequences of the present invention) at the same time during vector construction. 90% of the amount expressed in the presence of a nucleic acid sequence containing no Kozak and tbs sequences, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3% , 2%, 1%, 0.5% or 0.1%.

[0212] In the context of the present invention, the protein from the NOI at any given time during vector construction Expression of the target gene is overlapped (unlike the nucleic acid sequences of the present invention) at the same time during vector construction. 90% of the amount expressed in the presence of a nucleic acid sequence containing no Kozak and tbs sequences, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3% , 2%, 1%, 0.5% or even less than 0.1%.

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

[0214] Methods for analyzing and / or quantifying the translation of NOIs are well known in the art. do.

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

[0216] Improved Leader Sequence The TRIP system was tested using different promoters (containing different natural 5'UTRs of different lengths and compositions). When applied to a promoter, it maintains good levels of expression without TRAP. To confer efficient repression by TRAP, a tbs sequence was inserted within the promoter-UTR context. Since the start of this research, it has been hoped that TBS will be able to be applied in various configurations. Mediated by TRAP-tbs when inserted into the native UTR of a constitutive promoter It was not known what the achievable level of inhibition would be. To avoid any potential variability in repression levels that may be induced by the 5'UTR sequence of To achieve this, when modifying the selected promoter, a single conserved sequence is required along with the tbs. It would be advantageous to be able to provide a 5'UTR leader sequence. The first exon of the EF1α promoter (SEQ ID NO: 25) contains the natural leader sequence. Consistently better levels of transgene silencing by TRAP compared to 5'UTR leaders This leader also provides good levels of transgene expression in the absence of TRAP. It was found to provide

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

[0218] multiple cloning site To improve the tractability of the TRIP system, several different restriction enzymes (REs) were used. Through selection, N was inserted into an expression cassette containing the promoter-5'UTR-tbs sequence. The ability to directly clone OI, i.e., multiple cloning between tbs and Kozak sequences. It is desirable that the nucleotide sequence be capable of incorporating a multi-linking site (MCS) (see Figure 2A). The inventors have shown that several different MCSs can be tolerated by the TRIP system. Thus, it was demonstrated that transgene silencing can still be obtained when MCS is used. The 5'UTR leader sequence can regulate the degree of TRAP-mediated repression, tb The proximity of s to the ATG start codon is important and ensures efficient translation initiation. Therefore, an efficient Kozak sequence is required along with the start codon of the MCS and NOI. This was unexpected, given that the TRA The number and / or combination of RE sites that can be used while maintaining P-mediated repression is predicted. I couldn't do it.

[0219] Therefore, while maintaining the efficient core Kozak sequence of RVVATG, the AT Several (overlapping) RE sites were inserted from tbs to ATG (to maintain proximity to ATG). The sequence needs to be "compressed" so that it can be incorporated as close as possible to the start codon. was done.

[0220] In a further aspect, the present invention provides a method for combining a nucleotide of interest with a tb s or a portion thereof, a multiple cloning site (MCS), and a Kozak sequence as described herein. a nucleic acid sequence comprising a nucleotide sequence, the MCS being downstream of a tbs or a portion thereof and a Kozak sequence; The nucleic acid sequence is provided, which is located upstream of the sequence. Suitably, the nucleic acid sequence is a tbs or a part thereof and The ZACK sequence does not overlap.

[0221] As used herein, a "multiple cloning site" refers to a number of cloning sites located in close proximity to one another. It should be understood as a DNA region containing several restriction enzyme recognition sites (restriction enzyme sites). In one embodiment, 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" refers to a site that is D containing a specific sequence of nucleotides 4 to 8 nucleotides long that is recognized by A restriction enzyme recognizes a specific RE site (i.e., a specific sequence). and cleaves the DNA molecule within or near the RE site.

[0223] A consensus TRAP binding site sequence that can bind TRAP may bind multiple times (e.g., Repeated (e.g., 6, 7, 8, 9, 10, 11, 12 or more times) N], where K can be T or G in DNA and U in RNA. or G. In one embodiment of the present invention, the TRAP binding site or a portion thereof may be Column KAGN ≧2 (e.g., KAGN 2~3 ) Therefore, for the avoidance of doubt, This tbs or a portion thereof may contain, for example, the following repeat sequences: UAGNN, GAGNN, TA "N" means any of the following: GNN, UAGNNN, GAGNNN, or TAGNNN. It should be understood that any nucleotide may be designated at that position in the string. For example, can be G, A, T, C or U. The number of such nucleotides is preferably 2. but up to three, e.g., one, two, or three, and 11 x repeat tbs or a portion thereof KAG repeats can be separated by three spacing nucleotides, resulting in translational repression Preferably, the maximum amount of TRAP that results in translational repression is 100%. In order to retain the TRAP binding activity of the 11× repeat tbs or a portion thereof, one or more The lower N3 spacer will be used.

[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: where "K" can be T or G and "R" represents a purine at that position in the sequence (i.e. , A, or G), and "V" should be understood to designate any of G, A, or C. It should be understood that the term "nucleotide" designates any nucleotide.

[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 sequences of the invention comprise overlapping tbs-MCS-Kozak sequences. : GAGTTTAGCGGAGTGGAGAAGAGCGGAGCCGAGCCTAGC AGAGACGAGAAGAGCTCTAGACCATG (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 a viral vector produced by a viral vector producing cell, wherein translation of the NOI is It can interact with TRAP so that it is inhibited in

[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 includes any one of SEQ ID NOs: 8 to 19 or 22. It can be seen.

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

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

[0236] In one embodiment, tbs or a portion thereof is selected from the group consisting of the sequence KAGN 2~3 Contains 11 repetitions of Suitably, the number of KAGNNN iterations is 3 or less, e.g., tbs or The portion may include 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); , "R" is understood to designate a purine (i.e., A or G) at that position in the sequence. and "V" is understood to designate any nucleotide from G, A, or C. should be.

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

[0239] In one embodiment, the transcription start site / promoter ends at tbs or a portion thereof. The distance to the beginning is less than 34 nucleotides.

[0240] In one embodiment, the transcription start site / promoter ends at tbs or a portion thereof. The distance to the beginning is less than 13 nucleotides.

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

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

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

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

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

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

[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 located at the 3' end of the tbs or part thereof and downstream of the NOI. It is 3 or 9 nucleotides from the start codon.

[0250] In one embodiment, the spacer comprises a sequence defined in any one of SEQ ID NOs: 38 to 44. 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. Transcription of the motor results in the 5'UTR being encoded in the resulting mRNA transcript. Promoters are known in the art and are used to control the expression of a nucleotide of interest. The promoter can be any promoter suitable for carrying out the transcription of EF1α, EF2α, EF3α, EF4α, EF5α, EF6α, EF7α, EF8α, EF9α, EF10α, EF11α, EF12α, EF13α, EF14α, EF15α, EF16α, EF17α, EF18α, EF19α, EF20α, EF2 It can be S, CMV or CAG.

[0255] In a preferred embodiment, the overlapping tbs and Kozak sequences described herein are Located within the 5'UTR of the motor, the 5'UTR contains native sequences from the associated promoter. or more preferably, the 5'UTR may comprise the 5'UTR sequences described herein. It is composed.

[0256] In a preferred embodiment, a compressed / overlapped sequence is provided between the tbs and the Kozak sequence described herein. A sequence containing the overlapping MCS is located within the 5'UTR.

[0257] Overlapping tbs and Kozak sequences as described herein or tbs and The Kozak sequence contains a compressed / overlapping MCS between the 3' and 5' UTRs. It may be located at the end.

[0258] Preferably, the 5'UTR has the following sequences: SEQ ID NOs: 29-37, 45-58, 69-9 2 and one of SEQ ID NOs: 108 to 116. More preferably, the 5'UTR comprises one of SEQ ID NOs: 29 or SEQ ID NO: 108. Even more preferably, the 5'UTR comprises SEQ ID NO: 29. include.

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

[0260] The promoter is a promoter that is expressed in an intronless viral vector genome, e.g., CMV. The promoter may be any promoter typically used in the art.

[0261] In a preferred embodiment, the promoter-5'UTR region is an artificial promoter comprising a heterologous intron. Therefore, the promoter-5'UTR region has been engineered to contain heterologous exon-intron-exon sequences, The mature 5'UTR encoded within the A transcript is excised by intron splicing. The promoter-5'UTR sequence is generated using methods known in the art. For example, the promoter may be engineered as described herein. (See Example 8).

[0262] Preferably, it results from splicing out of an intron or a heterologous intron. The expression of transgene proteins from their mature mRNA is efficiently achieved by TRAP. Suitably, the intron or heterologous intron is as set forth in SEQ ID NO: 122. It may be an EF1α intron sequence.

[0263] The intron or heterologous intron may be the overlapping tbs and Kozak upstream of a sequence comprising an MCS between the tbs and the Kozak sequence of the sequence or described herein, That is, it may be located 5'.

[0264] The 5'UTR contained the following sequence (chicken β-actin / rabbit β-globin chimeric 5'UTR). TR - intron, bolded exon sequences (spliced ​​together, 5'UTR Become a leader)): CGGCGGGCGGGAACGTTGCCTTCGCCCCGTGCCCCGCTC CGCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCG CGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCC CTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTCGT TTCTTTTCTGTGGCTGCGTGAAAGCCTTAAAGGGCTCCGG GAGGGCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGT GCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCG CTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGC TTTGTGCGCTCCGCGTGTGCGCGAGGGGAGCGCGGGCCGG GGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGAACA AAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGG GGGTGTGGGCGCGGCGGTCGGGCTGTAACCCCCCCCTGGC ACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGG TGCGGGGCTCCGTGCGGGGCGTGGCGCGGGGCTCGCCGTG CCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGG CGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCG CGGCGGCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGA GCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGC GCAGGGACTTCCTTTGTCCCAAATCTGGCGGAGCCGAAAT CTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGCG AAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGG GCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCATCT CCAGCCTCGGGGCTGCCGCAGGGGGACGGCTGCCTTCGGG GGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGAC CGGCGGCTTTAGAGCCTCTGCTAACCATGTTCATGCCTTC TTCTTTTTCCTACAGCTCCTGGGCAAA (SEQ ID NO: 121) may include:

[0265] The 5'UTR is composed of the following sequence (EF1a5'UTR-intron, exon sequence in bold ( spliced ​​together to form the 5'UTR leader)). CTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGT GCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTT ATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCA GTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGG TGGGAGAGTTCGTGGCCTTGCGCTTAAGGAGCCCCTTCGC CTCGTGCTTGAGTTGTGGCCTGGCCTGGGCGCTGGGGCCG CCGCGTGCGAATCTGGTGGCACCTTCGCGCCCTGTCTCGCT GCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGAC CTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAA TGCGGGCCAAGATCAGCACACTGGTATTTCGGTTTTTGGG GCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACAT GTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAAT CGGACGGGGGTAGTCTCAAGCTGCCCGGCCTGCTCTGGTG CCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGG CAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAG ATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCACAAAATGG AGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCA CACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTC ATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTC GATTAGTTCTCCAGCTTTTGGAGTACGTCGTCTTTAGGTT GGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGA GTGGGTGGAGACTGAAGTTAGCCAGCTTGGCACTTGATG TAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTT GGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTT TCTTCCATTTCAGGTGTCGTGAAAA (SEQ ID NO: 122) may include:

[0266] In one embodiment, the promoter comprises the sequence (bold exon sequence (spliced ​​exon sequence) together, forming the 5'UTR leader), tbs consensus in italics): CGGCGGGCGGGAACGTTGCCTTCGCCCCGTGCCCCGCTC CGCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCG CGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCC CTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTCGT TTCTTTTCTGTGGCTGCGTGAAAGCCTTAAAGGGCTCCGG GAGGGCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGT GCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCG CTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGC TTTGTGCGCTCCGCGTGTGCGCGAGGGGAGCGCGGGCCGG GGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGAACA AAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGG GGGTGTGGGCGCGGCGGTCGGGCTGTAACCCCCCCCTGGC ACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGG TGCGGGGCTCCGTGCGGGGCGTGGCGCGGGGCTCGCCGTG CCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGG CGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCG CGGCGGCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGA GCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGC GCAGGGACTTCCTTTGTCCCAAATCTGGCGGAGCCGAAAT CTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGCG AAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGG GCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCATCT CCAGCCTCGGGGCTGCCGCAGGGGGACGGCTGCCTTCGGG GGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGAC CGGCGGCTTTAGAGCCTCTGCTAACCATGTTCATGCCTTC TTCTTTTTCCTACAGCTCCTGGGCAAA[KAGN 2-3 ] 10-1 1 (SEQ ID NO: 123) Includes.

[0267] In one embodiment, the promoter comprises the sequence (bold exon sequence (spliced ​​exon sequence) together, forming the 5'UTR leader), tbs consensus in italics): CTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGT GCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTT ATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCA GTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGG TGGGAGAGTTCGTGGCCTTGCGCTTAAGGAGCCCCTTCGC CTCGTGCTTGAGTTGTGGCCTGGCCTGGGCGCTGGGGCCG CCGCGTGCGAATCTGGTGGCACCTTCGCGCCCTGTCTCGCT GCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGAC CTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAA TGCGGGCCAAGATCAGCACACTGGTATTTCGGTTTTTGGG GCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACAT GTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAAT CGGACGGGGGTAGTCTCAAGCTGCCCGGCCTGCTCTGGTG CCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGG CAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAG ATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCACAAAATGG AGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCA CACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTC ATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTC GATTAGTTCTCCAGCTTTTGGAGTACGTCGTCTTTAGGTT GGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGA GTGGGTGGAGACTGAAGTTAGCCAGCTTGGCACTTGATG TAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTT GGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTT TCTTCCATTTCAGGTGTCGTGAAAA[KAGN 2-3 ] 10-11 ( SEQ ID NO: 124) Includes.

[0268] In one embodiment, the promoter is a chicken promoter having the sequence (tbs-kzkV0.G variant). Chicken β-actin / rabbit β-globin chimeric 5'UTR-intron, exons in bold Sequences (spliced ​​together to form the 5'UTR leader), tbs in italics kzkV0.G): CGGCGGGCGGGAACGTTGCCTTCGCCCCGTGCCCCGCTC CGCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCG CGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCC CTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTCGT TTCTTTTCTGTGGCTGCGTGAAAGCCTTAAAGGGCTCCGG GAGGGCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGT GCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCG CTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGC TTTGTGCGCTCCGCGTGTGCGCGAGGGGAGCGCGGGCCGG GGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGAACA AAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGG GGGTGTGGGCGCGGCGGTCGGGCTGTAACCCCCCCCTGGC ACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGG TGCGGGGCTCCGTGCGGGGCGTGGCGCGGGGCTCGCCGTG CCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGG CGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCG CGGCGGCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGA GCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGC GCAGGGACTTCCTTTGTCCCAAATCTGGCGGAGCCGAAAT CTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGCG AAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGG GCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCATCT CCAGCCTCGGGGCTGCCGCAGGGGGACGGCTGCCTTCGGG GGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGAC CGGCGGCTTTAGAGCCTCTGCTAACCATGTTCATGCCTTC TTCTTTTTCCTACAGCTCCTGGGCAAAGAGTTTAGCGGAG TGGAGAAGAGCGGAGCCGAGCCTAGCAGAGACGAGCCGAG ATG (SEQ ID NO: 117) Includes.

[0269] In one embodiment, the promoter comprises a sequence (having overlapping tbs and Kozak sequences) EF1a 5'UTR-intron (tbskzkV0.G variant), exon sequence in bold (spliced ​​together to form the 5'UTR leader), tbskz in italics kV0.G): CTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGT GCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTT ATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCA GTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGG TGGGAGAGTTCGTGGCCTTGCGCTTAAGGAGCCCCTTCGC CTCGTGCTTGAGTTGTGGCCTGGCCTGGGCGCTGGGGCCG CCGCGTGCGAATCTGGTGGCACCTTCGCGCCCTGTCTCGCT GCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGAC CTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAA TGCGGGCCAAGATCAGCACACTGGTATTTCGGTTTTTGGG GCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACAT GTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAAT CGGACGGGGGTAGTCTCAAGCTGCCCGGCCTGCTCTGGTG CCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGG CAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAG ATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCACAAAATGG AGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCA CACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTC ATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTC GATTAGTTCTCCAGCTTTTGGAGTACGTCGTCTTTAGGTT GGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGA GTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATG TAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTT GGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTT TCTTCCATTTCAGGTGTCGTGAAAAGAGTTTAGCGGAGTG GAGAAGAGCGGAGCCGAGCCTAGCAGAGACGAGCCGAGAT G (SEQ ID NO: 118) contains

[0270] Spliced ​​sequence corresponding to SEQ ID NO: 117; 5'UTR leader sequence in bold , italicized tbskzkV0.G: CGGCGGGCGGGAACGTTGCCTTCGCCCCGTGCCCCGCTC CGCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCG CGTTACTCCCACAGCTCCTGGGCAAAGAGTTTAGCGGAGT GGAGAAGAGCGGAGCCGAGCCTAGCAGAGACGAGCCGAGA TG (SEQ ID NO: 119) Spliced ​​sequence corresponding to SEQ ID NO: 118; 5'UTR leader sequence in bold , italicized tbskzkV0.G: CTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTGTCG TGAAAAGAGTTTAGCGGAGTGGAGAAGAGCGGAGCCGAGC CTAGCAGAGACGAGCCGAGATG (SEQ ID NO: 120) Exemplary Nucleic Acid Sequences Exemplary nucleic acid sequences of the invention are shown below.

[0271] Sequence number 62-L33 Improved reader, optimal (overlapping) tbs([KAGNN]8 Exemplary nucleic acid sequences containing 1)-Kozak junctions CTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGAGTTT AGCGGAGTGGAGAAGAGCGGAGCCGAGCCGAGATG Sequence number 63-L33 Improved reader, optimal (overlapping) tbs([KAGNN]1 1)-Exemplary nucleic acid sequences containing Kozak junctions 2 CTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGAGTTT AGCGGAGTGGAGAAGAGCGGAGCCGAGCCTAGCAGAGACG AGCCGAGATG Sequence number 64-L12 improved reader, optimal (overlapping) tbs([KAGNN]1 1)-Kozak-containing exemplary nucleic acid sequence 3 CTTTTTCGCAACGAGTTTAGCGGAGTGGAGAAGAGCGGA GCCGAGCCTAGCAGAGACGAGCCGAGATG SEQ ID NO: 65—Exemplary nucleic acid sequence for an intron-containing 5′ UTR 4, including L33 Spliced ​​leader, optimal (overlapping) tbs ([KAGNN] 11 )-Ko Bringing Zach Bonding CTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTGTCG TGAAAAGAGTTTAGCGGAGTGGAGAAGAGCGGAGCCGAGC CTAGCAGAGACGAGCCGAGATG SEQ ID NO: 66 - Improved spacer, optimal (overlapping) tbs([KAGNN]8) - Exemplary Nucleic Acid Sequences Containing Kozak Junctions 5 ATAGCAGAGACGGCTGAGTTTAGCGGAGTGGAGAAGAGC GGAGCCGAGCCGAGATG Sequence number 67-L33 Improved reader tbs([KAGNN] 11 )- MCS -Kozatt Exemplary nucleic acid sequences containing CTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGAGTTT AGCGGAGTGGAGAAGAGCGGAGCCGAGCCTAGCAGAGACG AGAA GAGCTCTAGA CCATG SEQ ID NO: 68 - Improved spacer, tbs([KAGNN] 11 )- MCS -Kozak Exemplary nucleic acid sequence 7 containing ATAGCAGAGACGGCTGAGTTTAGCGGAGTGGAGAAGAGC GGAGCCGAGCCTAGCAGAGACGAGAA GAGCTCTAGA CCAT G 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 to 36, preferably any one of SEQ ID NOs: 34 to 36 one of; or (ii) any one of SEQ ID NOs: 45 to 58, preferably any one of SEQ ID NOs: 52 to 58 t 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 to 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 to 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 present invention, the nucleotide of interest is expressed in a target cell lacking TRAP. It is translated in the following way.

[0278] "Target cells" should be understood as cells in which it is desired to express the NOI The NOI may be introduced into a target cell using the viral vector of the present invention. Delivery to cells can be 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 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 mutant of target protein, toxin, conditional toxin, antigen, transcription Factors, structural proteins, reporter proteins, intracellular localization signals, tumor suppressor proteins Proteins, growth factors, membrane proteins, receptors, vasoactive proteins and peptides, antivirals reporter proteins and ribozymes, and their derivatives (with associated reporter groups) The NOI includes, but is not limited to, sequences encoding the polypeptides of the present invention. Without wishing to be bound by theory, microRNAs may also encode The processing of this protein is thought to be 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 inhibits an enzyme or enzymes involved in dopamine synthesis. For example, the enzyme can be one or more of the following: tyrosine hydroxylase (TYH) hydroxylase, GTP-cyclohydrolase I and / or aromatic amino acid dopa dehydrogenase carboxylase. The sequences of all three genes are available (GenBa nk® accession numbers X05290, U19523 and M76180).

[0284] In another embodiment the NOI may encode vesicular monoamine transporter 2 (VMAT2) In an alternative embodiment, the viral genome encodes the aromatic amino acid dopa decarboxylase. Such a genome may comprise a NOI encoding a VMAT2 gene and a NOI encoding VMAT2. , 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 is a therapeutic protein or a combination of therapeutic proteins may be coded.

[0286] In another embodiment the NOI is a glial cell line derived neurotrophic factor (GDNF), a brain derived neurotrophic factor. Neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), neurotrophin-3 (N T-3), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF) ), interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), insulin VEGF-2, VEGF-A, VEGF-B, VEGF-C / VEGF-2, VEGF -D, VEGF-E, PDGF-A, PDGF-B, PDFG-A and PDFG-B One or more proteins selected from the group consisting of heterodimers and homodimers It can be coded.

[0287] In another embodiment the NOI is angiostatin, endostatin, platelet factor 4, color Physiological epithelium-derived factor (PEDF), placental growth factor, restin, interferon-α, interferon -feron-inducible protein, globatin- and tuberculosis-1, interleukin- (IL)-1, IL-12, retinoic acid, anti-VEGF antibodies or fragments / variants thereof, e.g. For example, aflibercept, thrombospondin, VEGF receptor proteins, e.g., U.S. Pat. Those described in U.S. Patent Nos. 5,952,199 and 6,100,071 and one anti-angiogenic protein selected from the group consisting of an anti-VEGF receptor antibody. Alternatively, it may encode multiple anti-angiogenic proteins.

[0288] In another embodiment the NOI comprises an NF-kB inhibitor, an IL1β inhibitor, a TGFβ inhibitor, IL-6 inhibitors, IL-23 inhibitors, IL-18 inhibitors, tumor necrosis factor alpha and tumor necrosis Factor β, lymphotoxin α and β, LIGHT inhibitor, α-synuclein inhibitors, tau inhibitors, beta amyloid inhibitors, and IL-17 inhibitors. The present invention may encode an anti-inflammatory protein, antibody, or fragment / variant of the protein or antibody. do.

[0289] In another embodiment the NOI encodes the cystic fibrosis transmembrane conductance regulator (CFTR). It can be coded.

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

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

[0292] In another embodiment the NOI is RPE65, aryl hydrocarbon interacting receptor protein Acetyltransferase-like 1 (AIPL1), CRB1, lecithin retinal acetyltransferase (L RAT), photoreceptor-specific homeobox (CRX), retinal guanylate cyclase ( cyclise) (GUCY2D), RPGR-interacting protein 1 (RPGRIP1) , LCA2, LCA3, LCA5, dystrophin, PRPH2, CNTF, ABCR / ABCA4, EMP1, TIMP3, MERTK, ELOVL4, MYO7A, USH2 A, VMD2, RLBP1, COX-2, FPR, Harmonin, Rab Escort Tampa quality 1, CNGB2, CNGA3, CEP290, RPGR, RS1, RP1, PREL P, encoding a protein selected from the group including glutathione pathway enzymes and opticin possible.

[0293] In another embodiment the NOI may encode human coagulation factor VIII or IX. do.

[0294] In another embodiment the NOI is a phenylalanine hydroxylase (PAH), methyl Malonyl-CoA mutase, propionyl-CoA carboxylase, isovaleryl-CoA decarboxylase Hydrogenase, branched-chain ketoacid dehydrogenase complex, glutaryl-CoA dehydrogenase acetyl-CoA carboxylase, propionyl-CoA carboxylase, 3-methyl Dihydrochloride-CoA carboxylase, pyruvate carboxylase, carbamoyl- Phosphate synthase, ammonia, ornithine transcarbamylase, glucosylceramide β-galactosidase, α-galactosidase A, glucosylceramidase β, cystinosin, glucosamine (N-acetyl)-6-sulfatase, N-acetyl-α-glucosaminidase, N- Sulfoglucosamine sulfohydrolase, galactosamine-6 ​​sulfatase, aryl Sulfatase A, cytochrome B-245β, ABCD1, ornithine carbamoyltransferase lyase, argininosuccinate synthase, argininosuccinate lyase (lys ase), arginase 1, alanine glycoxhylate amine Selected from the group including ATP-binding cassette, subfamily B members The gene may encode one or more proteins involved in metabolism, of which one or more are selected.

[0295] In other embodiments the NOI is a chimeric antigen receptor (CAR) or a T cell receptor (TC In one embodiment, the CAR is an anti-5T4 CAR. The NOIs are B cell maturation antigen (BCMA), CD19, CD22, CD20, and CD13. 8, CD30, CD33, CD123, CD70, prostate-specific membrane antigen (PSMA), Wheel Y antigen (LeY), tyrosine-protein kinase transmembrane receptor 1 (ROR1), whip Muc1, cell surface binding molecule (Muc1), epithelial cell adhesion molecule (EpCAM), endothelial growth factor receptor Body (EGFR), insulin, protein tyrosine phosphatase, non-receptor type 22 , interleukin 2 receptor α, helicase C domain 1-induced interferon Human epidermal growth factor receptor (HER2), glypican 3 (GPC3), disialogang GD2, mesiothelin, vesicular endothelial growth factor receptor It may encode VEGFR2.

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

[0297] In a further aspect the NOI is selected from the group consisting of SGSH, SUMF1, GAA, common gamma chain (CD1 32), adenosine deaminase, WAS protein, globin, α-galactosidase A , δ-aminolevulinic acid (ALA) synthase, δ-aminolevulinic acid dehydratase ( ALAD), hydroxymethylbilane (HMB) synthase, uroporphyrinogen ( URO) synthase, uroporphyrinogen (URO) decarboxylase, coproporphyrinogen (URO) synthase Protoporphyrinogen (COPRO) oxidase, protoporphyrinogen (PROTO ) oxidase, ferrochelatase, α-L-iduronidase, iduronate sulfatase Heparan sulfamidase, N-acetylglucosaminidase, heparan-α-glucosaminidase Cosaminide N-acetyltransferase, 3N-acetylglucosamine 6-sulfa tase, galactose-6-sulfatase, β-galactosidase, N-acetyl Galactosamine-4-sulfatase, β-glucuronidase and hyaluronidase It can be coded.

[0298] In addition to the NOI, the vector may also contain an siRNA, shRNA or regulated shRNA. It may contain or encode A. (Dickins et al. (2005) Na ture Genetics 37:1289-1295, Silva et al. 2005)Nature Genetics 37:1281-1288) Indications Vectors, including retroviral vectors and AAV vectors, according to the present invention are internationally recognized. International Publication No. 1998 / 05635, International Publication No. 1998 / 07859, International Publication No. 1998 One or more N-amino acids useful in the treatment of disorders listed in US Pat. No. 6,998,555. The nucleotide of interest can be DNA or RNA. Possibly. Examples of such disorders include: cytokine and cell proliferation / differentiation activity; immunosuppressive or immunostimulatory activity (e.g., human For the treatment of immune deficiencies, including infection with immunodeficiency viruses, modulation of lymphocyte proliferation; and many autoimmune diseases, as well as preventing transplant rejection or tumor inducing immunity); regulating hematopoiesis (e.g., treating myeloid or lymphoid disorders); bone, Promoting the growth of cartilage, tendons, ligaments and nerve tissue (e.g., in wound healing, burns, bruises, etc.) for the treatment of ulcers and periodontal disease and neurodegeneration); inhibition or activation of follicle-stimulating hormone (regulation of fertility); chemotactic / chemokinetic activity (e.g., damaging or infecting specific cell types) 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, thus anti-inflammatory activity; anti Immune activity (i.e., cellular and / or humoral immune responses, including responses not associated with inflammation) Inhibitory effect on macrophages and T cells on extracellular matrix components and fibroblasts Inhibition of the ability to adhere to fibronectin and upregulated Fas receptors in T cells Disorders that respond to condition manifestations.

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

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

[0301] Arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders Harm, respiratory distress syndrome, cardiovascular effects, peripheral vascular disease, migraine and aspirin-dependent antithrombotic effects Vascular diseases including cerebrovascular disease, 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] Phenylketonuria (PKU), Wilson's disease, organic acidemia, urea cycle disorders, cholestasis Inherited metabolic disorders, including urinary tract infections and other diseases.

[0305] Kidney and urinary tract 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 disorders, dermatitis or other skin disorders .

[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 disorders .

[0309] Placental dysfunction, placental insufficiency, habitual miscarriage, eclampsia, preeclampsia, endometriosis and other maternal diseases Gynecological diseases, including gynecological diseases.

[0310] Leber congenital amaurosis (LCA), including LCA10, posterior uveitis, and intermediate uveitis , anterior uveitis, conjunctivitis, chorioretinitis, retinal uveitis, optic neuritis, open-angle glaucoma, and glaucoma, including juvenile congenital glaucoma; intraocular inflammation, e.g., retinitis or cystoid macular edema; Sensitive ophthalmitis, scleritis, retinitis pigmentosa, age-related macular degeneration (AMD), Best's disease, Best's egg Macular degeneration including juvenile macular degeneration including macular degeneration, Stargardt disease, Usher syndrome Group, Doyne honeycomb retinal dystrophy, Sorby's macular dystrophy , juvenile retinoschisis, cone-rod dystrophy, corneal dystrophy, Fuchs' dystrophy Fee, Leber congenital amaurosis, Leber hereditary optic neuropathy (LHON), Adie syndrome, Oguchi disease, degenerative ocular fundus (fondus) disease, ocular trauma, ocular inflammation caused by infection, Proliferative vitreoretinopathy, acute ischemic optic neuropathy, e.g., excessive scarring after glaucoma filtration surgery, eye transplantation Reaction to corneal grafts, corneal graft rejection, and diabetic macular edema, retinal vein occlusion, and RLBP 1 Ophthalmology, including other eye diseases such as associated retinal dystrophies, total choroidal atrophy, and color vision disorders mental disorder.

[0311] Parkinson's disease, complications and / or side effects of Parkinson's disease treatment, AIDS Associated dementia complex HIV-associated encephalopathy, Devic's disease, Sydenham's chorea, Alzheimer's disease and Other degenerative diseases, conditions, or disorders of the CNS, stroke, post-polio syndrome, psychiatric disorders, myelitis, Encephalitis, subacute sclerosing panencephalitis, encephalomyelitis, acute neurological disorder, subacute neurological disorder, chronic neurological disorder, Fabry disease, Gaucher disease, cystinosis, Pompe disease, metachromatic leukodystrophy, Wisconsin-related Wiscott-Aldrich syndrome, adrenoleukodystrophy, beta-thalassemia Mia, sickle cell disease, Guillain-Barré syndrome, Sydenham chorea, severe Myasthenia gravis, pseudotumor cerebri, Down's syndrome, Huntington's disease, CNS compression or CNS trauma, or or CNS infections, muscular atrophies and dystrophies, diseases of the central and peripheral nervous system, conditions or disorders, amyotrophic lateral sclerosis, spinal muscular atrophy, spinal cord and motor neuromuscular disorders including avulsion injuries Neurological and neurodegenerative disorders, including rheumatoid arthritis.

[0312] Cystic fibrosis, Sanfilipo syndrome A, Sanfilipo syndrome B , Sanfilippo syndrome C, Sanfilippo syndrome D, Hunter syndrome, Hurler-Scheie syndrome syndrome, mucopolysaccharidoses including Morquio syndrome, adenosine deaminase-related severe combined immunodeficiency ( ADA-SCID), X-linked severe combined immunodeficiency, X-linked chronic granulomatous disease, porphyrin Hemophilia A, Hemophilia B, Post-traumatic inflammation, Hemorrhage, Coagulation and acute phase response, Cachexia, Anorexia , acute infection, septic shock, infectious disease, diabetes, complications or side effects of surgery, bone Bone marrow transplant or other transplant complications and / or side effects, cornea, bone marrow, organ, lens, pacemaker Manufacturers of natural or artificial cells, tissues and organs, such as natural or artificial skin tissue, Humoral and / or cellular therapy for the prevention and / or treatment of graft rejection in transplantation cases to suppress or inhibit the cytotoxic immune response, e.g., by infection with a virus carrier or A Other diseases and conditions, such as complications and side effects of gene therapy caused by IDS.

[0313] siRNA, microRNA and shRNA In certain other embodiments, the NOI comprises a microRNA. A very large group of small RNAs that are naturally produced in organisms, at least some of which are targets. It regulates the expression of target genes. It is the founding member of the microRNA family. The let-7 gene is a member of the worm's mitochondrial mitosis. Small, highly conserved proteins that regulate the expression of endogenous protein-coding genes during development The active RNA species is initially transcribed as a precursor of approximately 70 nt, It is processed post-transcriptionally to its mature approximately 21 nt form. Both -4 and -5 are hairpins that are processed into their mature forms by the Dicer enzyme. It is transcribed as an RNA precursor.

[0314] In addition to the NOI, the vector may also contain an siRNA, shRNA or regulated shRNA. A may contain or encode (Dickins et al. (2005) Nat ure Genetics 37:1289-1295,Silva et al.(2 005)Nature Genetics 37:1281-1288).

[0315] Double-stranded RNA (dsRNA)-mediated post-transcriptional gene silencing (PTG) Transposon (S) is a conserved cellular defense mechanism for controlling the expression of foreign genes. Random integration of elements such as DNA or viruses occurs by the synthesis of homologous single-stranded mRNA or This is thought to result in the expression of dsRNA that activates sequence-specific degradation of viral genomic RNA. This silencing effect is known as RNA interference (RNAi) (Ra lph et al. (2005) Nature Medicine 11:429-4 33) The RNAi mechanism is based on the synthesis of long dsRNA, approximately 21–25 nucleotides (nt) long RNA. These products are sequence-specific mediators of mRNA degradation. The resulting molecules are called small interfering RNAs or silencing RNAs (siRNAs). In mammalian cells, dsRNA longer than 30 bp activates the interferon response and It has been found to result in the cessation of protein synthesis and nonspecific mRNA degradation (St ark et al., Annu Rev Biochem 67:227-64(19 98) However, this response was not significantly reduced by using a 21-nt siRNA duplex. (Elbashir et al., EMBO J. Dec 3 ;20(23):6877-88(2001),Hutvagner et al.,S science.Aug 3,293(5531):834-8.Eupub Jul 1 2 (2001)), enabling the analysis of gene function in cultured mammalian cells. do.

[0316] NOIs and polynucleotides The polynucleotides of the present invention may comprise DNA or RNA. The polypeptide may be single-stranded or double-stranded. As a result of the degeneracy of the genetic code, there are many different polypeptides. It will be understood by those skilled in the art that a single oligonucleotide can encode the same polypeptide. Furthermore, using routine techniques, it will be possible to produce a polypeptide of the invention in To reflect the codon usage of any particular host organism in which the gene is to be expressed, one of skill in the art should Nucleotides that do not affect the polypeptide sequence encoded by the polynucleotides of the invention It is understood that leution substitutions may be made.

[0317] The polynucleotides may be modified by any method available in the art. Such modifications may be used to enhance the in vivo activity or lifespan of polynucleotides of the invention. can be performed.

[0318] Polynucleotides, such as DNA polynucleotides, may be produced recombinantly, synthetically, or by other suitable means. Polynucleotides such as DNA polynucleotides can be produced by any means available to the public. Nucleotides may also be cloned by standard techniques.

[0319] Using recombinant means, for example, polymerase chain reaction (PCR) cloning techniques Using this technique, longer polynucleotides will generally be generated. A primer set (e.g., about 15-30 nucleotides) adjacent to the target sequence desired to be matched. To generate primer pairs, primers are applied to mRNA or or cDNA, and performing PCR under conditions that result in amplification of the desired region. The amplified fragments are isolated (e.g., by purifying the reaction mixture on an agarose gel). The steps include isolating the amplified DNA and recovering the amplified DNA. Suitable restriction enzyme recognition so that the DNA can be cloned into a suitable vector The nucleotide sequence may be designed to contain the site.

[0320] Major splice donor RNA splicing is comprised of five small nuclear ribonucleoproteins (snRNPs). The process is catalyzed by a large RNA-protein complex called the spliceosome. The boundaries between introns and exons are determined by specific nucleotide sequences within the pre-mRNA These boundaries are called "splicing boundaries." The term "splice site" refers to the site at which the gene is cut and / or is identified by the eukaryotic splicing machinery as being suitable for joining to an alternative splice site. It refers to a polynucleotide that can be recognized by a specific sequence.

[0321] The splice sites allow for the excision of introns present in the pre-mRNA transcript. Typically, the 5' splice boundary is referred to as the "splice donor site" or "5' splice site." The 3' splice boundary is called the "splice acceptor site" or "3' splice acceptor site." Examples of splice sites include naturally occurring splices. splice sites, engineered or synthetic splice sites, canonical or consensus splices splice sites, and / or non-canonical splice sites, e.g., cryptic splice sites Examples include:

[0322] A splice acceptor site generally consists of three distinct sequence elements: the branch point or branch site , consisting of a polypyrimidine tract and an acceptor consensus sequence. Branching in eukaryotes The consensus sequence is YNYTRAC (where Y is a pyrimidine and N is any nucleoside). nucleotide and R is a purine). The sass sequence is YAG (where Y is pyrimidine) (e.g., Griffiths et al., t al., eds., Modern Genetic Analysis, 2nd e dition,WHFreeman and Company,New York( 2002). The 3' splice acceptor site is typically located at the 3' end of the intron. Located at the edge.

[0323] Therefore, the major splice donor site is a lentivirus for use in the present invention. The gene may be inactivated in the nucleotide sequence encoding the RNA genome of the vector.

[0324] In one aspect, the present invention provides a nucleic acid sequence according to the invention as described herein. The nucleic acid sequence is contained within the RNA genome of the lentiviral vector, The major splice donor site in the RNA genome of the target is inactivated, e.g., by mutation or Also provided are nucleic acid sequences in which the sequence is deleted or omitted.

[0325] In one aspect, the present invention provides a nucleic acid sequence according to the invention as described herein. The nucleic acid sequence is operably linked to the RNA genome of the lentiviral vector, The major splice donor site in the RNA genome of the tivirus vector is inactivated Also provided are nucleic acid sequences that have been, for example, mutated or deleted.

[0326] In one aspect, the nucleotide sequence encoding the RNA genome of the lentiviral vector a sequence of major splice donor sites in the RNA genome of the lentiviral vector; The nucleotide sequence is provided, in which the nucleotide sequence is inactivated, e.g., mutated or deleted. do.

[0327] The terms "canonical splice site" and "consensus splice site" are used interchangeably. It can be used interchangeably to refer to splice sites that are conserved among species.

[0328] 5' donor splice site and 3' splice site used in eukaryotic RNA splicing Consensus sequences for acceptor splice sites are well known in the art. These consensus sequences consist of a nearly invariant dinucleotide at each end of the intron: It contains 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 is AG / GTR (for DNA). AGT (where A is adenosine, T is thymine, G is guanine, and C is where R is a cytosine, R is a purine, and " / " indicates the cleavage site. Other constraints, such as secondary structure within the vRNA packaging region, may be imposed on the same sequence. It is well known in the art that in the genome of a mouse, the splice donor may deviate from this consensus. Non-canonical splice sites are also well known in the art. However, they occur rarely compared to the canonical splice donor consensus sequence.

[0330] A "major splice donor site" is typically a splice donor site located at the end of a viral vector nucleotide sequence. It is encoded within the native viral RNA packaging sequence located in the 5' region and integrated The first (dominant) splice in the viral vector genome This refers to the donor site.

[0331] In one aspect, the nucleotide sequence encoding the RNA genome of the lentiviral vector The sequence does not contain an active major splice donor site, i.e., splicing does not occur prior to splicing. does not originate from the major splice donor site in the nucleotide sequence, The splicing activity from this position is removed.

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

[0333] In the case of the HIV-1 virus, the major splice donor consensus sequence is (DNA where A is adenosine and T is thymine, G is guanine, C is cytosine, R is purine, and " / " indicates the cleavage site. .).

[0334] In one aspect of the present invention, the splice donor region, i.e., the major spliced ​​region prior to mutation, The region of the vector genome containing the nA site 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-M SD-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: 10 5-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). can form a stem-loop structure in the splice donor region of the vector genome. In one aspect, this sequence (SL2) is a nucleotide sequence according to the invention as described herein. It may be deleted from the sequence.

[0340] Thus, the present invention encompasses nucleotide sequences that do not contain SL2. Nucleotide sequences not including the sequence according to sequence number 102 are included.

[0341] In one aspect of the invention, the major splice donor site has 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 are , the following core sequence, " / " indicates the major splice donor and potential spliced ​​donors: The cleavage site at the ner site is: / GTGA / GTA (SEQ ID NO: 106).

[0343] In one aspect of the invention, the MSD-mutated vector genome contains a major splice donor. and at least two mutations in the potential splice donor "region" (SEQ ID NO: 106). and the first and second "GT" nucleotides are the major splice donor and and immediately 3' of the potential splice donor nucleotide.

[0344] In one aspect of the invention, the major splice donor consensus sequence is CTGGT (sequence The major splice donor site may contain the sequence CTGGT.

[0345] In one aspect, the nucleotide sequence is SEQ ID NO:9 prior to splice site inactivation. 4, 96, 97, 102, 103 and / or 106.

[0346] In one aspect, the nucleotide sequence is the nucleotide sequence of SEQ ID NO:94 unless inactivated. An inactivated nucleotide sequence with a cleavage site between nucleotides corresponding to bases 13 and 14 Contains a splice donor site.

[0347] In accordance with the invention described herein, the nucleotide sequence is an inactive cryptic spliced ​​fragment. In one aspect, the nucleotide sequence also contains a major splice donor site. does not contain an active cryptic splice donor site adjacent to (3' of) Splicing does not occur from the cryptic splice donor site, and the cryptic splice donor Splicing from the site has been removed.

[0348] The term "cryptic splice donor site" refers to a site that normally functions as a splice donor site. or the context of the flanking sequences (e.g., the presence of a nearby "preferred" splice donor). ) are used less efficiently as splice donor sites due to mutations in the adjacent sequences ( For example, mutations in nearby "preferred" splice donors can result in splices that function more efficiently. It refers to a nucleic acid sequence that can be activated to become a splice donor site.

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

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

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

[0352] In one aspect, the nucleotide sequence is the nucleotide sequence of SEQ ID NO:94 unless inactivated. Inactivated latent cleavage site between nucleotides corresponding to bases 17 and 18 It contains a target splice donor site.

[0353] In one aspect of the invention, the major splice donor site and / or adjacent cryptic splices are The splice donor site contains a "GT" motif. Both the splice donor site and the adjacent cryptic splice donor site were mutated. The mutated GT motif is located at 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. , 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] Inactivates the major splice donor site and adjacent cryptic splice donor sites To do this, a variety of different types of mutations can be introduced into the nucleic acid sequence.

[0362] In one aspect, the mutation eliminates or inhibits splicing activity in the splice region. The nucleotide sequences described herein are SEQ ID NOs: 94, 96 , 97, 102, 103 and / or 106 It may contain mutations or deletions.

[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 a nucleic acid sequence. The term "" refers to any change to a single nucleotide. Point mutations include, for example, deletions These include amino acid residues, transitions and transversions, which occur within protein coding sequences. If present, these are classified as nonsense, missense, or silent mutations. "Nonsense" mutations produce stop codons. "Missense" mutations produce stop codons. , which produces a codon that codes for a different amino acid. Codons that code for either the same amino acid or a different amino acid without changing function In a nucleic acid sequence containing a potential splice donor site, one or more Many point mutations can be introduced. For example, a nucleic acid sequence containing a cryptic splice site can be It can be mutated by introducing two or more point mutations into it. do.

[0365] To achieve attenuation of splicing from the splice donor region, the major splice At least several positions within the nucleic acid sequence containing the donor and potential splice donor sites are In one aspect, the mutations are introduced at the splice donor cleavage site. Canonical splice donor consensus sequence within four nucleotides at the break site In a matrix array, this is A 1 G 2 / G 3 T 4 and " / " is the cleavage site. Viruses where other constraints, such as secondary structures within the vRNA packaging region, are imposed on the same sequence In the genome, splice donor cleavage sites can deviate from this consensus. It is well known in the art. 3 T 4 Dinucleotides are generally classified as canonical nucleotides. The least variable sequence within the donor consensus sequence is G 3 and or T 4 It is well known that mutations to HI are most likely to achieve the greatest attenuation effect. For the major splice donor site in the V-1 viral vector genome, this is T 1 G 2 / G 3 T 4 and " / " is the cleavage site. For example, an HIV-1 viral vector For potential splice donor sites in the genome, this is G 1 A 2 / G 3 T 4 It could be , " / " is the cleavage site. Furthermore, point mutation(s) may occur in the splice donor region. For example, a point mutation can be introduced upstream or adjacent to the splice donor site. The major splice donor site and / or the cryptic splice donor site can be introduced downstream. A nucleic acid sequence containing a Rice donor site is mutated by introducing multiple point mutations into it. In certain embodiments, the point mutations are located upstream and / or downstream of the cryptic splice donor site. can be introduced into

[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, a mutant sequence may be prepared that is flanked by restriction sites that allow ligation to fragments of the native sequence. Mutations are introduced at specific loci by synthesizing oligonucleotides containing After ligation, the resulting reconstructed sequence contains the desired nucleotide insertion, substitution, or deletion. This includes derivatives having the formula:

[0367] Other known techniques that allow for alteration of DNA sequences include Gibson assembly, Gol Recombination approaches such as den-gate cloning and in-fusion can be.

[0368] Alternatively, the specific codons may be altered according to the desired substitution, deletion, or insertion. Oligonucleotide-directed site-specific (or segmented) gene transfer is used to provide the altered sequence. Mutagenesis procedures (site-specific) can be used. Deletion or truncation of splice site variants The construct may be constructed by utilizing convenient restriction endonuclease sites flanking the desired deletion. can also be constructed.

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

[0370] An exemplary method for making the above modifications is described in Sambrook et al. (Molecul ar cloning:A Laboratory Manual,2d Ed.,Co Spring Harbor Laboratory Press, 1989) Thus, it is disclosed.

[0371] Splice site variants were identified by PCR mutagenesis, chemical mutagenesis, and forced nucleotide mismatches. by convolution (e.g., Liao and Wise, 1990) or by randomly varying by using induced oligonucleotides (Horwitz et al., 1988 9) Chemical mutagenesis (Drinkwater and Klinedinst, 1986) ) technology.

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

[0373] Combination with modified U1 Reduced ability to participate in aberrant splicing events both during LV production and in target cells Therefore, lentiviral vectors mutated with MSD are promising candidates for gene therapy vectors. However, they are preferred over current standard lentiviral vectors for use in Until the present invention, the production of vectors mutated with MSD was limited to the HIV-1 tat protein ( have relied on the supply of (first and second generation lentiviral vectors) or (third generation vectors) (in vectors) due to the non-stabilizing effect of mutating the MSD on vector RNA levels. For safety reasons, tat has been "reintroduced" into the current third generation LV system. The introduction of these drugs is not desirable or justified, and as a result, there is currently no clinical use intended. There is no solution to the reduced titer produced by MSD-mutated vectors shown.

[0374] The present inventors have developed a method for producing a vector genome RNA that binds to the 5' packaging region of the vector genome RNA during production. Co-expression of modified U1 snRNA, which directs the expression of MSD, resulted in the production of mutated third-generation This shows that tat-independent (i.e., Tat-independent) LVs can be produced to high titers. These modified U1snRNAs are associated with the presence of a 5' polyA signal within the 5'R region. It was shown that MSD-mutated LV production titers can be enhanced in an independent manner, and poly Other uses of modified U1 snRNA to suppress adenylation (so-called U1 interference) Surprisingly, the modified U1snR Targeting NA to a critical sequence in the packaging region increased the titer of LV with mutated MSD. The present inventors have demonstrated that the greatest enhancement of the titer of LV mutated with MSD occurs. Such MSDs are less pronounced and are due to the modified U1 snRNA. Within the major splice donor region, where the potency enhancement of the mutated LV variant is greatest. Novel sequence variations of the .

[0375] The inventors have surprisingly found that the endogenous sequence (splice donor site) is no longer targeted. Instead, it is engineered to target sequences within the vRNA molecule, based on U1 snRNA. Co-expression of a non-coding RNA that encodes the lentiviral vector enhances its output. As demonstrated in the Examples, the present inventors have found that the efficacy of the method can be enhanced. The modified U1 snRNA (major splice donor and cryptic splice donor) Lentiviruses harboring attenuating mutations within the major splice donor region (containing the donor site) The relative enhancement of the output titer of vectors containing the unmutated major splice donor region was observed. This shows that the IgG1A1 gene is larger than the standard lentiviral vector.

[0376] As demonstrated in the Examples, reduced titers within the major splice donor region Vectors with a wide range of mutation types (point mutations, region deletions, and sequence replacements) that result The genome can be used in combination with modified U1 snRNA. This approach Co-expression of the modified U1 snRNA with other vector components during vector production The modified U1 snRNA may correspond to a target sequence in the vector genome vRNA. Binding to consensus splice donor sites by replacing them with complementary heterologous sequences The present invention is based on the target sequence and complementary length, design and expression. Describes various application modes and optimal features of modified U1 snRNA, including modes .

[0377] In one aspect of the invention described herein, the vector comprises a modified U1snR It may be used in combination with NA, which is discussed further below.

[0378] Splicing and polyadenylation are important processes, especially for most protein-coding transcripts. In higher eukaryotes, where the mRNA contains multiple introns, it is an important step for mRNA maturation. Elements within the pre-mRNA required for splicing include the 5' splice It contains the splice donor signal, the sequence surrounding the branch point, and the 3' splice acceptor signal. These three elements interact with the spliceosome, which is involved in the synthesis of U1snRN. Five small nuclear RNAs (snRNAs) and associated nuclear proteins (snRNPs) containing A U1 snRNA is expressed by the polymerase II promoter. and is present in most eukaryotic cells (Lund et al., 1984, J. Biology ol. Chem., 259: 2013-2021). Human U1 snRNA (small nuclear R NA) is 164 nt long and has a well-defined structure consisting of four stem-loops. (West, S., 2012, Biochemical Society Trans U1 snRNA contains an exon at its 5' end. It contains a short sequence broadly complementary to the 5' splice donor site of the intron-intron junction. U1 snRNA forms a splice site by base pairing to the 5' splice donor site. Involved in selection and spliceosome assembly. U1snRN outside of splicing The known function of A is in regulating 3' end mRNA processing, and the initial poly(A) signal. suppresses premature polyadenylation (polyA) of

[0379] Human U1 snRNA (small nuclear RNA) is 164 nt long and contains four stem-loop sequences. It has a well-defined structure consisting of an endogenous non-coding RNA, U1 snRNAs are naturally occurring splice donor anneals during the early stages of intron splicing. Consensus 5' splice sequences (e.g., 5'-ACUUACCUG-3') are inserted via Rice donor site (e.g., 5'-MAGGURR-3', where M is A or C) Stem loop I is important for polyA repression. Stem loop II binds to the U1A-70K protein, which has been shown to be a key regulator of U1A-70K. It binds to protein A, and the 5'-AUUUGUGG-3' sequence, along with stem-loop IV, It binds to Sm proteins, which are important for U1 snRNA processing. The modified U1 snRNA for use in accordance with the present invention is a naturally occurring U1 snRNA, as defined in The vector genome vRNA fragment is inserted at the site of the splice donor targeting / annealing sequence. The vector has been modified to introduce a heterologous sequence complementary to a target sequence within the vector (see Figure 1).

[0380] As used herein, "modified U1snRNA," "redirected U1sn RNA," "retargeted U1snRNA," "repurposed U1snRNA," and and "mutant U1 snRNA" are the RNAs that initiate the splicing process of target genes. The consensus 5' splice donor site sequence (e.g., 5'-MAGG) used for U1 snRNA is a modified U1 snRNA that is no longer complementary to the U1 snRNA (URR-3'). Therefore, the modified U1 snRNA contains a splice donor site sequence (e.g., 5'-MA Instead, the U1 snRNA is modified so that it is no longer complementary to the U1 snRNA (GGURR-3'). Furthermore, the modified U1 snRNA is a mutated MSD in the lentiviral vector genome. A nucleotide sequence containing a unique RNA sequence (target site) within the packaging region of the molecule , i.e., targeting or complementary to sequences unrelated to vRNA splicing. The MSD is designed to be a package of mutated lentiviral vector genome molecules. The nucleotide sequence within the cleavage region can be preselected. The U1snRNA was expressed as a lentiviral vector genome with its 5' end mutated into MSD. U1 modified to be complementary to a nucleotide sequence within the packaging region of the HIV-1 molecule As a result, without wishing to be bound by theory, modified The U1 snRNA is modified to have a short sequence at the 5' end of the U1 snRNA and a target site sequence. It binds to the target site sequence based on complementarity, thereby stabilizing the vRNA and inhibiting the MSD. The mutated lentiviral vectors are believed to result in increased output vector titers. .

[0381] As used herein, "native splice donor annealing sequence" and "native splice donor annealing sequence" refer to The term "splice donor targeting sequence" refers to the consensus 5' splice donor sequence of an intron. It refers to a short sequence at the 5' end of endogenous U1 snRNA that is broadly complementary to the splice donor site. The natural splice donor annealing sequence is 5'-ACUUACCUG-3'. could be.

[0382] As used herein, the term "consensus 5' splice donor site" refers to a , e.g., having the sequence 5'-MAGGURR-3', This refers to the consensus RNA sequence at the 5' end of an intron.

[0383] As used herein, "MSD" refers to a lentiviral vector genome sequence that has been mutated. The "nucleotide sequence within the packaging region of the target gene" and the "target sequence" are also referred to as "target site." The term was previously used to refer to the target site for binding / annealing of the modified U1 snRNA. The packaging region of the selected MSD-mutated lentiviral vector genome molecule It refers to a site that has a specific RNA sequence within it.

[0384] As used herein, "MSD-mutated lentiviral vector genome molecule" refers to a lentiviral vector having a mutated MSD. Lentiviral vector genome sequence with mutated "packaging region" and "MSD" The term "packaging region" refers to the region from the beginning of the 5' U5 domain to the gag gene. The 5'-terminal region of the lentiviral vector genome is mutated to the end of the MSD sequence. Therefore, the MSD is a region of the lentiviral vector genome molecule that has been mutated. The packaging region consists of a 5'U5 domain, a PBS element, and a stem-loop (SL)1 element. element, SL2 element, SL3ψ element, SL4 element and gag gene It contains sequences derived from the host. It is not feasible to provide the complete gag gene in trans into the genome during antiviral vector production. Nucleotide sequence of the gag gene provided in trans. does not have to be encoded by the wild-type nucleotide and can be codon-optimized; important The main attributes of the gag gene provided in trans are gag and gagpol The purpose of lentiviral vectors is to encode and direct the expression of proteins. If the complete gag gene is provided in trans during vector production, it is called a "lentiviral vector." The term "packaging region of the target genome molecule" refers to the region from the beginning of the 5' U5 domain to the SL Lentiviruses with mutated MSDs up to the "core" packaging signal of the 3ψ element The 5'-terminal region of the vector genome molecule and the ATG codon (present within SL4) The natural gag sequence from the end of the remaining gag nucleotide sequence present on the vector genome It will be understood by those skilled in the art that the term "g" may refer to a g nucleotide sequence.

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

[0386] As used herein, "U1, which includes the natural splice donor annealing sequence" refers to a Introducing heterologous sequences within the first 11 nucleotides of snRNA, "Introducing the heterologous sequence within one nucleotide of the 5' end of U1 snRNA" and " The term "introduction of a heterologous sequence within the first 11 nucleotides of U1 snRNA" refers to the first 11 nucleotides or all or part of the 9 nucleotides from positions 3 to 11 of with the heterologous sequence, or by substituting U1s to have the same sequence as the heterologous sequence. Modify the first 11 nucleotides of the mRNA or the 9 nucleotides from positions 3 to 11 This includes:

[0387] As used herein, "inclusion of a heterologous sequence within a natural splice donor annealing sequence" refers to "Introducing" and "annealing of the natural splice donor at the 5' end of U1 snRNA The term "introducing a heterologous sequence into a sequence" refers to the natural splice donor annealing sequence. Replacing all or part of a sequence with the heterologous sequence, or a sequence identical to the heterologous sequence. The method includes modifying the natural splice donor annealing sequence to have:

[0388] As used herein, the term "enhancing lentiviral vector titer" refers to "Increase lentiviral vector titer", "Restore lentiviral vector titer" "Enables" and "improves lentiviral vector titer."

[0389] Thus, in one embodiment, the modified U1 snRNA is a mutant version of the MSD. The vector is designed to bind to a nucleotide sequence within the packaging region of the viral vector genome sequence. It has been modified as follows.

[0390] In some embodiments, the modified U1 snRNA is a lentivirus with a mutated MSD. a heterologous sequence complementary to a nucleotide sequence within the packaging region of the virus vector genome. To do so, it is modified at the 5' end compared to the endogenous U1 snRNA.

[0391] In some embodiments, the modified U1 snRNA is a lentivirus with a mutated MSD. a heterologous sequence complementary to a nucleotide sequence within the packaging region of the virus vector genome. To introduce the natural splice donor into the annealing sequence, we compared it with the endogenous U1 snRNA. It is modified at the 5' end in comparison.

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

[0393] The modified U1 snRNA may be a modified U1 snRNA variant. Therefore, the U1 snRNA variant to be modified is a naturally occurring U1 snRNA variant, U1 U1snRN containing a mutation within the stem-loop I region that eliminates -70K protein binding A variant, or contains a mutation in the stem-loop II region that eliminates U1A protein binding The U1 snRNA may be a stem cell-specific mutant that ablates U1-70K protein binding. U1 snRNA variants containing mutations within the loop I region are designated U1_m1 or U1_m2, Preferably, it may be U1A_m1 or U1A_m2.

[0394] In some embodiments, the modified U1 snRNA described herein is The major U1 snRNA sequence [cloverleaf] (nt 410-5) of the U1_256 sequence described 62) and at least 70% identity (appropriately at least 75%, at least 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 In some embodiments, the modifications of the present invention include nucleotide sequences having a nucleotide sequence identity of 1 to 100%. The U1 snRNA identified is the major U1 snRNA sequence of the U1_256 sequence described herein. Contains the cloverleaf sequence (nt 410-562). The RNA sequence [cloverleaf] (nt 410 to 562) is as follows:

[0395] GCAGGGGAGATACCATGATCACGAAGGTGGTTTCCCAG GGCGAGGCTTATCCATTGCACTCCGGATGTGCTGACCCCT GCGATTTCCCCAAATGTGGGAAACTCGACTGCATAATTTG TGGTAGTGGGGGACTGCGTTCGCGCTTTCCCCTG. (SEQ ID NO: 131) In some preferred embodiments, the natural splice donor annealing sequence is included. The first 11 nucleotides of U1 snRNA were mutated to MSD in a lentiviral vector. - A heterologous sequence complementary to a nucleotide sequence in the packaging region of the genome, in whole or in part Suitably, the first 11 nucleotides 1 to 11 ( Suitably, 2 to 11, 3 to 11, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 ) nucleic acid in the packaging region of the lentiviral vector genome in which the MSD has been mutated The nucleotide sequence is replaced with a heterologous sequence complementary to the nucleotide sequence.

[0396] In some embodiments, the natural splice donor annealing sequence is a mutated MSD. complementary to a nucleotide sequence within the packaging region of the lentiviral vector genome The native splice donor amino acid sequence may be replaced in whole or in part by a heterologous sequence. Ring arrangement 1-11 (appropriately 2-11, 3-11, 5-11, 1, 2, 3, 4, 5 6, 7, 8, 9, 10, or 11) a lentiviral vector in which the nucleic acid is mutated to MSD - nucleotide sequences within the packaging region of the genome are replaced with heterologous sequences complementary to the nucleotide sequences In a preferred embodiment, the entire natural splice donor annealing sequence does not alter the MSD. The nucleotide sequence within the packaging region of the lentiviral vector genome was The natural splice donor annealing sequence ( For example, 5'-ACUUACCUG-3') can be completely replaced with a heterologous sequence as described herein. can be obtained.

[0397] In some embodiments, the MSD is packaged in a mutated lentiviral vector genome. A heterologous sequence complementary to a nucleotide sequence within the ligation region is In some embodiments, the MSD comprises at least 7 nucleotides complementary to the The nucleotide sequence within the packaging region of the lentiviral vector genome was The complementary heterologous sequence has at least 9 nucleotides complementary to the nucleotide sequence. Preferably, the heterologous sequence for use in the present invention comprises a sequence corresponding to said nucleotide sequence. It contains 15 nucleotides complementary to

[0398] Suitably, the heterologous sequence for use in the present invention comprises between 7 and 25 (suitably between 7 and 20 , 7~15, 9~15, 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 MSD is packaged in a mutated lentiviral vector genome. The nucleotide sequence within the aging region includes the 5'U5 domain, the PBS element, and the SL1 element. element, SL2 element, SL3ψ element, SL4 element and / or g It is located within a sequence derived from the ag gene. Suitably, the MSD is mutated into a lentivirus The nucleotide sequences within the packaging region of the vector genome are SL1, SL2 and / or SL3. or located within the SL3ψ element. In some preferred embodiments, the MSD is The nucleotide sequence within the packaging region of the lentiviral vector genome that was altered was: Located within the SL1 and / or SL2 elements. The MSD was mutated by inserting a nucleotide sequence within the packaging region of the lentiviral vector genome. The nucleotide sequence is located within the SL1 element.

[0401] In some embodiments, the MSD is packaged in a mutated lentiviral vector genome. The nucleotide sequence within the cleavage region contains at least seven nucleotides. In this embodiment, the MSD is a mutated lentiviral vector genome packaging region. The nucleotide sequence within the region comprises at least 9 nucleotides. Suitably, the MSD The nucleotide sequence within the packaging region of the mutated lentiviral vector genome is , 7-25 (appropriately 7-20, 7-15, 9-15, 7, 8, 9, 10, 11, 12 , 13, 14 or 15) nucleotides.

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

[0403] Nucleotidyl transfer of nucleoside into the packaging region of the lentiviral vector genome with mutated MSD Binding of the modified U1 snRNA described herein to the peptide sequence is Compared to lentiviral vector production in the absence of modified U1 snRNA, lentiviral vector production in the absence of modified U1 snRNA This can enhance the lentiviral vector titer during viral vector production. The production of lentiviral vectors in the presence of modified U1 snRNA as described herein can be achieved by: Lentiviral Vector Production in the Absence of the Modified U1 snRNA Described Herein The lentiviral vector titer is enhanced compared to the lentiviral vector titer obtained by the method of Example 1. Measurement of lentiviral vector titer Suitable assays for are as described herein. Suitably, lentivirus The production of the vector involves the use of the gag, env, rev and leucine sequences of the modified U1 snRNA. Co-expression of vector components including the RNA genome of an antiviral vector. The RNA genome of the virus vector can be the MSD-2KO RNA genome. In embodiments, the enhanced lentiviral vector titer is due to the presence of a functional 5' LTR polyA site. In some embodiments, the modified U1 of the present invention snRNA-mediated enhancement of lentiviral vector titers is due to the presence of vector genome fragments. It is independent of polyA site suppression in the 5'LTR.

[0404] In some embodiments, the MSD is packaged in a mutated lentiviral vector genome. Binding of the modified U1 snRNA described herein to a nucleotide sequence within the binding region In this case, the lentiviral vector in the absence of the modified U1 snRNA described herein The lentiviral vector titer during lentiviral vector production is lower than that during lentiviral vector production. The MSD may be increased by at least 30%. Suitably, the MSD may be mutated into a lentiviral vector genome. a modified U1snR as described herein to a nucleotide sequence within the packaging region of NA binding to mutated MSD in the absence of the modified U1snRNA described herein The MSD during production was compared with that of the mutated lentiviral vector. Increase the vector titer to at least 35% (or, more appropriately, at least 40%, 45%, 50%, 60%, 70%, 100%, 150%, 200%, 250%, 300%, 350%, 40 0%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 80 0%, 850%, 900%, 950%, 1,000%, 2,000%, 5,000%, or or 10,000%).

[0405] The modified U1 snRNA described herein comprises: (a) a modified U1 snRNA binding site; The MSD was mutated to bind to the packaging region of the lentiviral vector genome. (b) selecting a target site (a preselected nucleotide site) of U1sn The natural splice donor annealing sequence at the 5' end of the RNA (e.g., 5'-ACUUAC CUG-3') at the preselected nucleotide site selected in step (a) It can be engineered by introducing complementary heterologous sequences.

[0406] Using conventional techniques in molecular biology, the natural sequence at the 5' end of the endogenous U1 snRNA was isolated. within the price donor annealing sequence (e.g., 5'-ACUUACCUG-3') or Alternatively, it is within the ability of one skilled in the art to introduce complementary heterologous sequences into the target site. Generally speaking, suitable routine methods include substitution by directed mutagenesis or homologous recombination. Conversion is included.

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

[0408] The modified U1 snRNA described herein can be prepared according to methods generally known in the art. For example, the modified U1 snRNA can be produced by chemical synthesis or recombinant It can also be produced by DNA / RNA technology.

[0409] In one aspect, the nucleotide sequence encoding the modified U1 snRNA is different The nucleotide sequences may be present on different plasmids, for example.

[0410] Using conventional molecular and cell biology techniques, the modified U1snR described herein can be Introducing a nucleotide sequence encoding NA into a cell is well within the capabilities of one skilled in the art. be.

[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 movement of an entity from one environment to another. According to the present invention, for example, some of the vectors used in recombinant nucleic acid technology are A vector can be used to carry a segment of nucleic acid (e.g., a heterologous DNA segment, such as a heterologous cDNA segment). Vectors allow the introduction of entities such as nucleotides (e.g., nucleotides) into target cells. The purpose of preserving the seed nucleic acid (DNA or RNA) or the DNA or RNA segregation Replication of vectors containing the segment or proteins encoded by the nucleic acid segment This can serve the purpose of promoting the expression of quality.

[0413] The vectors of the present invention may, for example, be provided with an origin of replication, allowing expression of the polynucleotide. The virus may be provided with a promoter for expression and a regulator of the promoter. The vector may be a vector carrying one or more selectable marker genes. a gene (e.g., neomycin resistance gene) and / or a traceable marker gene (e.g., The vector may contain, for example, a gene encoding GFP. These may be used to induce and / or transduce

[0414] The vectors of the present invention may be used to replicate the NOI in compatible target cells in vitro. Thus, the present invention provides a method for transfecting a vector of the present invention into a compatible target cell. and growing the target cells under conditions that result in in vitro transfection and expression of the NOI. The present invention provides a method for producing proteins in vitro by The target cells may be harvested by methods well known in the art. Suitable target cells include mammalian Animal cell lines and other eukaryotic cell lines are included.

[0415] The vector can be an expression vector. The expression vectors described herein can be transcribed It includes the region of nucleic acid that contains the sequence. Therefore, mRNA, tRNA and rRNA are Sequences encoding the vector are included within this definition. Preferably, the expression vector is A polynucleotide of the invention operably linked to a control sequence capable of providing expression of the coding sequence. Contains nucleotides.

[0416] viral vectors In one embodiment of the present invention, the vector is a viral vector. It may also be called a vector, vector virion, or vector particle.

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

[0418] In one embodiment the viral vector comprises more than one NOI and at least one wherein the NOI is operably linked to a tbs or portion thereof as described herein.

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

[0420] It is anticipated that the suppression system of the present invention will be beneficial for any viral vector system. This system allows the nucleotide of interest to be delivered to, for example, a viral vector producing cell or It will find particular use in causing deleterious effects 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 an HIV-1, HIV-2, SIV, F IV, BIV, EIAV, CAEV or Visna lentivirus derived.

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

[0425] Retroviral and lentiviral vectors The retroviral vectors of the present invention can be derived from any suitable retrovirus, and The retrovirus may be derived from any suitable retrovirus. A number of different retroviruses are Examples include murine leukemia virus (MLV), human T-cell leukemia virus (HCV), and 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). For a detailed list, see Coffin et al. (1997) "Retroviruses" '',Cold Spring Harbor Laboratory Press Eds: JM Coffin, SM Hughes, HE 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 are known to cause cancer. The remaining two groups are lentiviruses and spiroviruses. A review of these retroviruses is given by Coffin et al. (1997) ibid. is presented in.

[0427] The basic structure of retroviral and lentiviral genomes is the genome packaging packaging signal, primer binding site, and an integration site that allows integration into the ga gene, as well as a ga gene encoding packaging components g / pol and env genes - These are polypeptides required for the assembly of virus particles. Many common genes, such as the 5'LTR and 3'LTR, are located between or within Lentiviruses share common features. They share the rev gene and RRE sequence of HIV. of integrated proviral RNA transcripts from the nucleus to the cytoplasm of infected target cells. It has additional features that allow for efficient unloading.

[0428] In the provirus, these genes are bound at both ends by long terminal repeats (LTRs). The LTRs are responsible for the integration and transcription of the provirus. The LTR also functions as an enhancer-promoter sequence, controlling the expression of viral genes. It can be controlled.

[0429] The LTR itself is an identical file that can be divided into three elements called U3, R, and U5. The U3 is derived from a sequence specific to the 3' end of the RNA. The R is a repeating sequence at both ends of the RNA. U5 is derived from a sequence unique to the 5' end of the RNA. The size of can vary considerably between different retroviruses.

[0430] In a typical retroviral vector of the present invention, one or more genes essential for replication are At least a portion of the more protein coding region can be removed from the virus. For example, gag / pol and env may not exist or may not function. This renders the viral vector replication-deficient.

[0431] Target non-dividing cells can be transduced and / or their genomes can be integrated into the host genome

[0039] Creating a vector containing a candidate nucleic acid binding sequence as described herein that can be incorporated into For this purpose, part of the viral genome also contains regulatory control regions and reporter sequences within the vector genome. - a library encoding the candidate nucleic acid binding sequences described herein operably linked to a gene May be replaced by Lee.

[0432] Lentiviruses are part of the larger group of retroviruses. For a detailed list, see Coffin et al. (1997) "Retroviruses" es'',Cold Spring Harbor Laboratory Pres s Eds: JM Coffin, SM Hughes, HE Varmus pp 7 58-763). Briefly, lentiviruses have been shown to be effective in infecting both primate and non-primate species. Examples of primate lentiviruses include those that cause human autoimmune deficiency syndromes (HIV). Human immunodeficiency virus (HIV), the causative agent of AIDS, and simian immunodeficiency Non-primate lentiviruses include, but are not limited to, SIV. In addition to the prototypical "slow virus," Visna / Maedi virus (VMV), there are related Caprine arthritis and encephalitis virus (CAEV), equine infectious anemia virus (EIAV), and feline immunodeficiency virus (FEV) These include the feline immunodeficiency virus (FIV) and the bovine immunodeficiency virus (BIV).

[0433] The Lentiviridae family is a group of viruses that have the ability to infect both dividing and non-dividing cells. They differ from retroviruses in that they possess the ability to EMBO J11(8):3053-3058 and Lewis and Emerma n(1994)J Virol 68(1):510-516). In contrast, MLV et al. Other retroviruses infect non-segmented organisms, such as the cells that make up muscle, brain, lung, and liver tissue. They are unable to infect cleft or slowly dividing cells.

[0434] Lentiviral vector, as used herein, refers to a vector derived from a lentivirus. Preferably, the component part is a vector containing at least one component part. It is involved in the biological mechanisms by which a virus infects cells, expresses genes, or replicates.

[0435] Lentiviral vectors can be derived from primate lentiviruses (e.g., HIV-1) or non-primate lentiviruses. The vector may be derived from any of the long-chain lentiviruses.

[0436] Examples of non-primate lentiviruses include any of the Lentiviridae family that do not naturally infect primates. Members may include feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), , Caprine Arthritis Encephalitis Virus (CAEV), Maedi-Visna Virus (MVV) or Equine It may include infectious anemia virus (EIAV).

[0437] Generally speaking, a typical retroviral vector production system requires the essential viral packaging. This involves the separation of the viral genome from the genomic functions. These components are typically separated into separate D NA expression cassette (also known as plasmid, expression plasmid, DNA construct or expression construct) The recombinant vector is provided to the production cells on a vector (known as a construct).

[0438] The vector genome comprises an NOI. The vector genome is typically signal (ψ), central polypurine tract (cppt), rev response element (RRE) NOI It requires an internal expression cassette with a post-transcriptional element (PRE), typically a central promoter. Requires liprin strand (cppt), 3'-ppu, and self-inactivating (SIN) LTR The R-U5 region is responsible for the correct transcription of both the vector genome RNA and the NOI mRNA. The vector genome is required for the processes of polyadenylation and reverse transcription. The open reading frame is It may include.

[0439] In one aspect, the nucleotide sequence is used in a tat-independent system for vector production. As described herein, the third generation lentiviral vectors may be suitable for use in The lentiviral vector is tat-independent and the nucleotide sequence of the present invention In one aspect of the present invention, the present invention can be used in the context of third generation lentiviral vectors. Therefore, tat is not provided in the lentiviral vector production system, e.g., tat is expressed in trans. In one aspect, the cells or vectors or vector products described herein are not provided. The live system does not contain tat protein.

[0440] The packaging functions include the gag / pol and env genes. These functions are required for the production of vector particles by producer cells. Providing the gene in trans facilitates the production of replication-defective viruses.

[0441] Production systems for gammaretroviral vectors typically contain the genome, gag / po It is a three-component system requiring the l and env expression constructs. The production system for the tiviral vector further comprises the accessory gene rev in trans. To be provided, the vector genome must contain a rev response element (RRE). EIAV-based lentiviral vectors contain an open reading frame (O If RF is present, rev is not required (see WO 2003 / 064665). see).

[0442] Typically, an "external" promoter (e.g., a promoter encoded within the vector genome cassette) is used. a promoter (driving the NOI cassette) and an "internal" promoter (driving the NOI cassette) All of these promoters, as well as those driving other vector system components, must be strong eukaryotic or bacterial promoters. Examples of such promoters include CMV, EF1α, Examples include the PGK, CAG, TK, SV40, and ubiquitin promoters. Strong "synthetic" promoters, such as those produced by libraries (e.g., the JeT promoter) A promoter may also be used to drive transcription. Rhodopsin (Rho), rhodopsin kinase (RhoK), cone-rod homeobox containing gene (CRX), neural retina-specific leucine zipper protein (NRL), yolk-like Macular dystrophy 2 (VMD2), tyrosine hydroxylase, neurospecific Enolase (NSE) promoter, astrocyte-specific glial fibrillary acidic protein (GF AP) promoter, human α1-antitrypsin (hAAT) promoter, phosphoe Pyruvate carboxykinase (PEPCK), liver fatty acid binding protein promoter promoter, Flt-1 promoter, INF-β promoter, Mb promoter, SP- B promoter, SYN1 promoter, WASP promoter, SV40 / hAlb promoter promoter, SV40 / CD43, SV40 / CD45, NSE / RU5' promoter , ICAM-2 promoter, GPIIb promoter, GFAP promoter, Fib Ironectin promoter, endoglin promoter, elastase-1 promoter, Desmin promoter, CD68 promoter, CD14 promoter and B29 promoter Tissue-specific promoters such as motors can be used.

[0443] Retroviral vector production involves transient transfection of producer cells with these DNA components. or stable producer cell lines (PCs) in which the components are integrated into the producer cell genome. L) (e.g., Stewart, HJ, MALerou x-Carlucci, CJSion, KAMitrophanous and P .A.Radcliffe(2009)Gene Ther.16(6):805-81 4 Epub 2009 Mar 2005). Another approach is to Stable packaging cells (in which the encoding components have been stably incorporated) are used, and then the required The virus of the present invention is transiently transfected into a vector genome plasmid according to the method. To generate a vector, the producer cell must be capable of expressing TRAP. Thus, in one embodiment of the present invention, the production cells stably express the TRAP construct. In another embodiment of the invention, the production cell transiently expresses the TRAP construct. In another embodiment of the invention, the production cell stably expresses the TRAP construct and Transiently express the P construct.

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

[0445] In one embodiment of the present invention, the viral vector is derived from EIAV. It has the simplest genome structure of any tivirus and is particularly preferred for use in the present invention. In addition to the ol and env genes, EIAV expresses three other genes: tat, rev, and Tat encodes S2. Tat acts as a transcriptional activator of the viral LTR (Derse and Newbold (1993) Virology 194(2):530-53 6 and Maury et al (1994) Virology 200(2):632 -642), rev regulates viral gene expression via the rev response element (RRE). Regulate and harmonize (Martarano et al. (1994) J Virol 68(5):3102-3111). The mechanism of action of these two proteins is It is widely believed to be similar to a similar mechanism in viruses (Martarano et al. (1994) J Virol 68(5):3102-3111). S2's Its function is unknown. Furthermore, it is spliced ​​into the env coding sequence at the beginning of the transmembrane protein. The EIAV protein Ttm, encoded by the first exon of tat, is In an alternative embodiment of the present invention, the viral vector is derived from HIV: HIV differs from EIAV in that it does not encode S2, but unlike EIAV, it encodes vi f, vpr, vpu and nef.

[0446] The term "recombinant retroviral or lentiviral vector" (RRV) refers to a vector In the presence of packaging components, RNA is packaged into viral particles that can infect target cells. A vector containing sufficient retroviral genetic information to allow packaging of the A genome. Infection of the target cell involves reverse transcription and integration into the target cell genome. RRVs carry non-viral coding sequences to be delivered to target cells by vectors. RRVs undergo independent replication to produce infectious retroviral particles within target cells. RRVs usually lack functional gag / pol and / or en They lack the v gene 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" refers to a nucleotide sequence of interest. Conferring the functions required to infect, transduce, and deliver the nucleotide sequence to target cells Viral vectors can be engineered to remove non-essential elements while retaining elements essential for the Further details of this strategy are provided in WO 1998 / 178 15 and WO 99 / 32646. The tar lacks the tat, rev, and S2 genes, none of which are required for the production system. The minimal HIV vector is vif, vpr, vpu, t Lacking at and nef.

[0449] However, the expression plasmid used to produce the vector genome in the producer cell The retroviral vector directs the transcription of the genome in the producer / packaging cells. The gene will contain transcriptional regulatory control sequences operably linked to the genome. These regulatory sequences include: may be the natural sequence associated with the transcribed retroviral sequence, i.e., the 5' U3 region, Alternatively, as described below, a different viral promoter, such as a CMV promoter, may be used. Some lentiviral vector genomes can be efficiently expressed as promoters. For example, in the case of HIV, the RRE sequence is included. However, the need for RRE (and the dependency on rev provided in transformer) The genetic diversity can be reduced or eliminated by codon optimization. Further details of this strategy are available in The same function as the rev / RRE system can be found in the International Publication No. 2001 / 79518. Other sequences that fulfill this role are known. For example, functional analogs of the rev / RRE system include the Masson-Franklin sequence. It is found in the Fazarian monkey virus as a constitutive transport element (CTE). RRE-type sequences in the genome that are known and thought to interact with factors in infected cells The cellular factors can be thought of as analogs of rev. Thus, the CTE contains the r Any other known or available system may be used as an alternative to the ev / RRE system. Functional equivalents may be suitable for the present invention. For example, the Rex protein of HTLV-I is It is also known that it can functionally replace the Rev protein of IV-1. v and RRE may be absent in vectors for use in the methods of the present invention. may be non-functional or non-functional, alternative rev and RRE, or functionally equivalent Such a system may exist.

[0450] SIN vector The vectors for use in the methods of the present invention preferably contain a viral enhancer and used in a self-inactivating (SIN) construct in which the promoter sequence is deleted. SIN vectors were constructed and expressed in vivo, ex vivo, or in vivo with efficacy similar to that of wild-type vectors. or can transduce non-dividing target cells in vitro. Inactivation of transcription of the long terminal repeat (LTR) should prevent vRNA recruitment, This is a feature that further reduces the likelihood of the formation of a virulent virus. Regulation of genes from internal promoters by eliminating any cis-acting effects This should also enable expression of

[0451] For example, self-inactivating retroviral vector systems utilize transcriptional elements within the U3 region of the 3'LTR. Constructed by deleting the enhancer or enhancer and promoter After a series of vector reverse transcription and integration, these changes were mediated by the 5' and 3' LTT R replicates in both the host and the host, producing a transcriptionally inactive "provirus." Any promoter(s) within the LTR in such a vector may still This strategy allows for the suppression of transcription from internally located genes. It has been used to eliminate the influence of enhancers and promoters in viral LTRs. Such effects include increased transcription or repression of transcription. This strategy It can also be used to eliminate downstream transcription from the LTR into genomic DNA. This is particularly true in human gene therapy, where it is important to prevent the accidental activation of endogenous oncogenes. This is of concern. Yu et al., (1986) PNAS 83:3194-98; Ma rty et al.,(1990)Biochimie 72:885-7;Navi aux et al.,(1996)J.Virol.70:5701-5;Iwaku ma et al.,(1999)Virol.261:120-32;Deglon et al.,(2000)Human Gene Therapy 11:179-9 0. SIN lentiviral vectors are disclosed in U.S. Patent No. 6,924,123 and U.S. Patent No. No. 7,056,699.

[0452] Non-replicating lentiviral vectors In the genome of replication-deficient lentiviral vectors, gag / pol and / or e The sequence of nv may be mutated, absent, and / or non-functional.

[0453] In a typical lentiviral vector of the present invention, a target gene essential for viral replication is At least a portion of the coding region of one or more of the proteins is removed from the vector. This makes the viral vector replication-deficient and allows it to transduce non-dividing target cells. and / or its genome can be integrated into the genome of a target cell. To generate a vector containing an NOI, a portion of the viral genome is inserted into the vector containing the nucleotide of interest. It can also be replaced by NOI.

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

[0455] In a further embodiment, the vector delivers sequences that lack or lack viral RNA. In a further embodiment, the method is capable of ensuring packaging of the RNA to be delivered. To achieve this, a heterologous binding domain (for gag) is located on the RNA to be delivered. heterologous) and the cognate binding domain on Gag or GagPol can be used. Both of these vectors are described in WO 2007 / 072056.

[0456] Adenovirus vectors In another embodiment of the present invention, the vector may be an adenoviral vector. Viruses are double-stranded, linear DNA viruses that do not replicate through an RNA intermediate. There are over 50 types of adenoviruses, divided into six subgroups based on their genetic sequence. There are different human serotypes.

[0457] Adenoviruses are capable of in vivo and ex vivo expression in a wide range of cell types of human and non-human origin. A non-enveloped double-stranded DNA virus capable of transduction in vivo and in vitro These cells include airway epithelial cells, hepatocytes, muscle cells, cardiac myocytes, synovial cells, and primary These include post-mitotically terminally differentiated cells such as mammary epithelial cells and neural cells.

[0458] Adenoviral vectors can also transduce non-dividing cells, such as the lung epithelium. This is crucial for diseases such as cystic fibrosis, where affected cells within the nucleus have a slow turnover rate. Indeed, the cystic fibrosis transporter (CFTR) is secreted into the lungs of affected adult cystic fibrosis patients. Several trials are underway using adenovirus-mediated delivery of HIV-1.

[0459] Adenoviruses are used as vectors for gene therapy and expression of heterologous genes. The large (36 kb) genome can accommodate up to 8 kb of foreign inserted DNA. and replicate efficiently in complementing cell lines, producing up to 10 12 Very high transducing units / ml Therefore, adenoviruses can produce high titers in primary non-replicating cells. It is one of the best systems for studying gene expression.

[0460] Expression of viral or foreign genes from the adenoviral genome is essential for replicating cells. Adenoviral vectors do not require endocytic cells. Once inside the cell, the adenoviral vector integrates into the host chromosome. Instead, adenoviral vectors are directly integrated into the host nucleus. It functions episomally (independently of the host genome) as a strand genome.

[0461] Adeno-associated virus vector Adeno-associated viruses (AAV) have a high integration frequency and are capable of infecting non-dividing cells. This makes it an attractive vector system for use in the present invention. Adeno-associated viruses (AAV) are useful for delivering genes into mammalian cells. V has a broad host range for infection. For further details, see U.S. Pat. Nos. 5,139,941 and 5,139,941, each of which is incorporated herein by reference. and U.S. Pat. No. 4,797,368.

[0462] Recombinant AAV vectors are useful for in vitro transcription of marker genes and genes involved in human diseases. It has been successfully used for ex vivo and in vivo transduction.

[0463] Certain AAV vectors can efficiently integrate large payloads (up to 8–9 kb) One such vector is a hybrid of AAV5 capsid and AAV2 ITR(Allocca M,et al J.Clin Invest(2008) 118:1955-1964).

[0464] Herpes simplex virus vector Herpes simplex virus (HSV) is an enveloped virus that naturally infects nerve cells. Herpes simplex virus (HSV) is a double-stranded DNA virus that carries large strands of foreign DNA. Herpes simplex virus (HSV) is a vector system that can house multiple compartments. and have been used as vectors for gene delivery to neural cells ( Manservigiet et al Open Virol J. (2010)4: 123-156).

[0465] The use of HSV in therapeutic procedures is important as the strain is unable to establish a lytic cycle. In particular, it is necessary to attenuate the HSV strain for use in human gene therapy. When used for the purpose of transfection, the polynucleotide should preferably be inserted into an essential gene. This is because when a viral vector encounters a wild-type virus, it recombines to produce the wild-type virus. This is because the introduction of foreign genes into the virus can occur. As long as the insert is in an essential gene, this recombination will not result in the loss of the target gene in the recipient virus. Essential genes are also deleted, allowing the "escape" of foreign genes into the replication-competent wild-type virus population. This will prevent

[0466] Vaccinia virus vector The vector of the present invention is a vaccinia virus vector, such as MVA or NYVAC. An alternative to vaccinia vectors is the fowlpox vector known as ALVAC. or canarypox vectors and infect and recombine in human cells. Strains derived from these that can express proteins but cannot replicate are listed. It can be obtained.

[0467] Baculovirus vectors The vector of the present invention may also be a baculovirus vector. Modification of baculovirus to allow expression of an encoded NOI has been well documented in the art. This can be achieved, for example, by the use of a mammalian promoter upstream of the NOI. It is possible.

[0468] Vectors encoding multiple NOIs In one embodiment the vector comprises more than one NOI and one or more wherein the NOI is operably linked to a tbs or portion thereof as described herein.

[0469] Internal ribosome entry site (IRES) As mentioned above, a vector of the invention may contain more than one NOI. To express I, two or more transcription units, each of which is a NO, may be included in the vector genome. However, there may be one for I. Retroviral vectors are genetically simple. If maintained, retroviral vectors have the highest titers and most potent gene expression characteristics. It is clear from the literature that the efficacy of ll et al.,1988 J.Virol.62,2464;Correll e t al.,1994 Blood 84,1812;Emerman and Tem in 1984 Cell 39,459;Ghattas et al.,1991 Mol.Cell.Biol.11,5848;Hantzopoulos et al .,1989 PNAS 86,3519;Hatzoglou et al.,199 1 J.Biol.Chem 266,8416;Hatzoglou et al., 1988 J.Biol.Chem 263,17798;Li et al.,199 2 Hum.Gen.Ther.3,381;McLachlin et al.,19 93 Virol.195,1;Overell et al.,1988 Mol.C ell Biol.8,1803;Scharfman et al.,1991 PN AS 88,4626;Vile et al.,1994 Gene Ther 1, 307;Xu et al.,1989 Virol.171,331;Yee et al. al., 1987 PNAS 84, 5197), and therefore polycistronic messages Intra-sequence ribosome progression occurs to initiate translation of the second (and subsequent) coding sequence in the sequence. It is preferable to use an IRES (Insertion Resistor Entry Site) (Adam et al 1991 J. Virol.65,4985).

[0470] Insertion of an IRES element into a retroviral vector inhibits the retroviral replication sequence. It is compatible with the nucleotide sequence, allowing expression of multiple coding regions from a single promoter ( Adam et al (above); Koo et al (1992) Virology 1 86:669-675;Chen et al 1993 J. Virol 67:21 42-2148). IRES elements are first found in the untranslated 5' ends of picornaviruses. where IRES elements are found to regulate cap-independent translation of viral proteins. (Jang et al (1990) Enzyme 44:292-309) When located between open reading frames in an RNA, an IRES element is , which promotes ribosome entry at the IRES element and subsequently the initiation of downstream translation. By translating the downstream open reading frame, .

[0471] A review of IRES is provided by Mountford and Smith (TIG May 1, 2013). 995 vol 11, No 5:179-184). Virus (EMCV) (Ghattas, IR, et al., Mol. Cell. Biol., 11:5848-5859(1991); BiP protein [Maceja k and Sarnow,Nature 353:91(1991)];Drosop hila antennapedia gene (exons d and e) [Oh, et al., Ge nes & Development, 6:1643-1653 (1992)] Others in poliovirus (PV) [Pelletier and Sonenber g,Nature 334:320-325(1988);Mountford and Smith, TIG 11, 179-184 (1985) IRES sequences are known.

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

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

[0474] The IRES(s) may be of viral origin (EMCV IRES (SEQ ID NO: 59) ), PV IRES or FMDV 2A-like sequences) or cellular origin (FGF 2 I RES, NRF IRES, Notch 2 IRES or EIF4 IRES) It could be.

[0475] To enable the IRES to initiate translation of each polynucleotide, ES is located between or before the polynucleotides in the vector genome. should be.

[0476] promoter Expression of the NOI is controlled by a regulatory sequence comprising a promoter / enhancer and other expression control signals. The promoter sequence can be controlled using a prokaryotic promoter and a promoter functional in eukaryotic cells. Tissue-specific or stimulus-specific promoters can be used. It can be used to express a sequence containing elements from two or more different promoters. Chimeric promoters may also be used.

[0477] Suitable promoter sequences include those from polyomavirus, adenovirus, fowlpox virus, bovine papillomavirus, and the like. sarcoma virus, avian sarcoma virus, cytomegalovirus (CMV), retroviruses, and and those derived from the genomes of viruses such as simian virus 40 (SV40), or ubiquitin promoter, EF1α, CAG, TK, SV40, ubiquitin, PGK or including those derived from heterologous mammalian promoters such as ubiquitous protein promoters It is a strong promoter. Alternatively, to drive transcription, rhodopsin (Rho), Rhodopsin kinase (RhoK), cone-rod homeobox-containing gene (CRX), neuronal Retina-specific leucine zipper protein (NRL), vitelliform macular dystrophy 2 (VMD2) D2), tyrosine hydroxylase, neurospecific enolase (NSE) promoter Astrocyte-specific glial fibrillary acidic protein (GFAP) promoter, human α l-antitrypsin (hAAT) promoter, phosphoenolpyruvate carboxyl kinase (PEPCK), liver fatty acid binding protein promoter, Flt-1 promoter promoter, INF-β promoter, Mb promoter, SP-B promoter, SYN1 promoter promoter, WASP promoter, SV40 / hAlb promoter, SV40 / CD 43, SV40 / CD45, NSE / RU5' promoter, ICAM-2 promoter , GPIIb promoter, GFAP promoter, fibronectin promoter, Endoglin promoter, elastase-1 promoter, desmin promoter, CD Tissue-specific promoters such as the 68 promoter, CD14 promoter, and B29 promoter A motor can be used.

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

[0479] The promoter may be selected to ensure or increase expression in the appropriate target cells. For example, the features may include conserved regions, e.g., Pri A promoter may be a nucleotide sequence consisting of a nucleotide sequence of a nucleotide sequence selected from the group consisting of a nucleotide sequence ... to affect (maintain, enhance, or decrease) the level of expression of the 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. Also, appropriate elements for enhancing transcription or translation may be present.

[0480] Constitutive and / or strong promoters driving the transgene, including tissue-specific promoters A promoter is desirable, especially when expression of the transgene protein in the production cell is dependent on the vector. resulting in reduced viral titers and / or increased production of transgene-derived proteins from the viral vector. TRAP-tbs interaction is essential for inducing immune responses in vivo due to target delivery. This formed the basis for a transgene protein silencing system for the production of retroviral vectors. This can be useful in creating

[0481] Regulators of NOIs Generation of retroviral packaging / producer cell lines and retroviral vector expression Complicating factors in target production include the specific retroviral vector components and NOI. Constitutive expression of these components is cytotoxic, resulting in the death of cells expressing these components. Therefore, the inability to produce these components (e.g., ga It can regulate the expression of proteins such as g-pol and envelope proteins like VSV-G. Expression of other non-cytotoxic vector components, such as rev, also places a metabolic burden on the cell. Therefore, the vector components described herein can be adjusted to minimize The modular construct or nucleotide sequence encoding the Cytotoxic and / or non-cytotoxic vector components with at least one regulatory element As used herein, the term "regulatory element" refers to a gene that is associated with Any gene or protein that can affect, increase or decrease its expression Regulatory elements include gene switch systems, transcriptional regulatory elements, and It contains a translational repression element.

[0482] Many prokaryotic regulator systems have been used to generate gene switches in mammalian cells. Many retroviral packaging and producer cell lines have been developed. Gene switch systems (e.g., tetracycline and cumate-inducible switches) This allows the retroviral vector to be regulated during vector production. The expression of one or more of the gene switcher components can be switched on. The TetR family of transcriptional regulators (e.g., T-Rex, Tet- On and Tet-Off gene switch systems, cumate-induced switches of transcriptional regulators gene switch systems (e.g., CymR proteins) and RNA-binding proteins Gene switch systems involving transcription factors (e.g., TRAP) are included.

[0483] One such tetracycline-inducible system is based on the T-REx™ system. For example, in such a system, The tetracycline operator (TetO2) is a nucleotide sequence of the human cytomegalovirus (HCV) The last nucleotide of the TATATAA element of the viral major immediate-early promoter (hCMVp) The nucleotide is 10 bp from the 3' end, and then TetR acts alone as a repressor. (Yao F, Svensjo T, W inkler T, Lu M, Eriksson C, Eriksson E.,199 8, Hum Gene Ther;9:1939-1950). In such a system, NO Expression of I is driven by a CMV promoter with two copies of the TetO2 sequence inserted in tandem. The TetR homodimer can be regulated by an inducer (tetracycline or other In the absence of doxycycline (dox), an analog of TetO2, it binds to the TetO2 sequence and When present, the inducer physically blocks transcription from the CMV promoter. The TetR homodimer binds to the TetO2 sequence, preventing the TetR homodimer from binding to the TetO2 sequence. Codon optimization improves translation efficiency by causing a steroidal change and resulting in gene expression. This was found to result in tighter control of TetO2-regulated gene expression. Thus, the TetR gene can be codon-optimized.

[0484] The TRiP system is described in WO 2015 / 092440 and is used to generate vectors. This provides an alternative method for suppressing expression of the NOI in the producing cells during the transcription process. Constitutive and / or strong promoters driving the transgene are preferred, including promoters In particular, expression of the transgene protein in the producer cells may result in a decrease in vector titer. and / or viral vector delivery of transgene-derived proteins. When eliciting an immune response in vivo, the TRAP-binding sequence (e.g., TRAP-tbs) The interaction is directed to a transgene protein suppression system for the production of retroviral vectors. Forming the basis (Maunder et al., Nat Commun. (2017) M ar 27;8).

[0485] Briefly, the TRAP-tbs interaction forms a translation block, leading to the transgene protein (Maunder et al., Nat Commun. (2017) Mar 27;8). The translation block is only effective in the producing cells, and therefore It does not interfere with DNA or RNA-based vector systems. The TRiP system Proteins are constitutively expressed containing tissue-specific promoters derived from mono- or bicistronic mRNAs. and / or can repress translation when expressed from a strong promoter. Unregulated expression of the transgene protein reduces vector titers and increases the vector product It has been demonstrated that toxicity or molecular burden can affect the quality of The problem of lar burden can lead to cell stress; Viral vector delivery of the protein allows the transgene protein to induce an immune response in vivo. If the use of gene editing transgenes induces on-target / off-target effects; transgene protein may have adverse effects on the vector and / or or transient and stable, if they may affect the clearance of envelope glycoproteins. The suppression of transgene protein for both PaCL / PCL vector production systems was The cells are beneficial to prevent a drop in vector titer.

[0486] Packaging Sequence When used within the context of this invention, it is interchangeable with "packaging sequence" or "psi." The term "packaging signal" as used in the literature refers to the signal that is transmitted to the retrovirus during viral particle formation. Used for non-coding cis-acting sequences required for encapsidation of viral RNA strands. In HIV-1, this sequence is located upstream of the major splice donor site (SD) and It has been mapped to a locus extending at least to the gag initiation codon. In the present study, the packaging signal contains an R region in the 5' coding region of Gag.

[0487] As used herein, an "extended packaging signal" or "extended packaging signal" refers to a The term "gag sequence" refers to the sequence surrounding the psi sequence with further extensions in the gag gene. The inclusion of these additional packaging sequences allows for the production of This can increase the efficiency of vector RNA insertion into the host.

[0488] Feline immunodeficiency virus (FIV) RNA encapsidation determinants are separate and non-contiguous and one region (R-U5) at the 5' end of the genomic mRNA and the proximal 311 region of gag. It has been shown that the region contains another region that has been mapped within nt (Kaye et al. 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, HIV The env gene product of the base vector is expressed by cells that express a protein called CD4. However, the env If the gene 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 pseudotyped an HIV-based vector with a glycoprotein derived from VSV (Verm a and Somia(1997)Nature 389(6648):239-24 2).

[0490] In another alternative, the Env protein is a mutant or engineered Env protein. The modifications may be to introduce targeting capabilities or may be administered or selected to reduce toxicity or other purposes (Val sesia-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 the references cited therein).

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

[0492] VSV-G The envelope glycoprotein of vesicular stomatitis virus (VSV), a rhabdovirus ( G) is a pseudotype of certain enveloped viruses and viral vector virions. It is an envelope protein that has been shown to be capable of being cytosine-modified.

[0493] MoMLV-based retroviral vectors in the absence of any retroviral envelope proteins Its ability to pseudotype viral vectors was demonstrated by Emi et al. (1991) Jour nal of Virology 65:1202-1207) WO 1994 / 294440 reports that retroviral vectors have been successfully used with VSV-G. These pseudotyped VSV-G vectors are It can be used to transduce a wide range of mammalian cells. More recently, Abe et al. al.(1998)J Virol 72(8)6356-6361, VSV-G It teaches that the addition of a non-infectious retroviral particle can make it infectious. do.

[0494] Burns et al.(1993)Proc.Natl.Acad.Sci.US A 90:8033-7) successfully pseudotyped the retrovirus MLV with VSV-G, This resulted in a vector with an altered host range compared to its native MLV form. VSV-G pseudotyped vectors can be expressed in mammalian cells as well as in fish, reptiles, and insects. It has also been shown to infect cell lines derived from worms (Burns et al. (1993) ibid.). VSV-G pseudotyped vectors express conventional amphotropic envelope vectors in a variety of cell lines. It has also been shown to be more efficient than rope (Yee et al., 1994 )Proc.Natl.Acad.Sci.USA 91:9564-9568,Emi et al. (1991) Journal of Virology 65:1202 The VSV-G protein interacts with the retroviral core through its cytoplasmic tail. can act as a vector and can be used to pseudotype specific retroviruses. Cut.

[0495] Providing pseudotyped envelopes of non-retroviruses, such as VSV-G protein, This offers the advantage of allowing vector particles to be concentrated to high titers without loss of infectivity (Akk ina et al. (1996) J. Virol. 70:2581-5). Perhaps Because it consists of two non-covalently linked subunits, the retroviral envelope The loop proteins are apparently unable to withstand the shear forces during ultracentrifugation. The interaction between the glycoproteins can be disrupted by centrifugation. Therefore, VSV-G protein pseudotyping has potential benefits. Points can be provided.

[0496] WO 2000 / 52188 describes a membrane-bound viral envelope protein. Stable producer of vesicular stomatitis virus G protein (VSV-G) We describe the generation of pseudotyped retroviral vectors from cell lines, and It provides high-quality gene sequences.

[0497] Ross River virus The Ross River virus envelope is a non-primate lentiviral vector (FIV). It has been used to pseudotype and primarily transduced the liver after systemic administration (Kang et al. t al(2002)J Virol 76(18):9378-9388). The efficiency is 20-fold greater than that obtained with vectors pseudotyped with VSV-G, suggesting hepatotoxicity It caused less cytotoxicity as measured by serum levels of liver enzymes. It was reported that:

[0498] Baculovirus GP64 The baculovirus GP64 protein is required for clinical and commercial use. Regarding viral vectors used for large-scale production of high-titer viruses, alternatives to VSV-G have been proposed. It has been shown that it is a substitute (Kumar M, Bradow BP, Zimmerb erg J (2003) Hum Gene Ther. 14(1):67-77). VS Compared with the VG pseudotyped vector, the GP64 pseudotyped vector showed similar It has broad tropism and similar natural titer. GP64 expression does not kill cells, so A 293T-based cell line can be generated that constitutively expresses GP64.

[0499] Alternative Envelopes Other envelopes that give reasonable titers when used with pseudotyped EIAV include Mokola , rabies, Ebola, and LCMV (lymphocytic choriomeningitis virus).407 Intravenous injection of 0A pseudotyped lentivirus into mice resulted in maximum genetic expression in the liver. This resulted in the development of a newborn.

[0500] Viral vector production systems and cells Another aspect of the invention is a method for producing a viral vector comprising: A viral vector production system comprising a set of nucleic acid sequences, wherein the vector genome sequence is a nucleic acid sequence of the present invention. The present invention relates to a viral vector production system comprising a nucleic acid sequence.

[0501] A "viral vector production system" or "vector production system" or "production system" means a virus It should be understood as a system containing the components necessary for vector production.

[0502] Therefore, vector production systems contain the components necessary to generate viral vector particles. One such nucleic acid sequence is a set of nucleic acid sequences encoding a gene encoding TRAP. In a preferred embodiment, the RNA binding protein is a bacterial TRAP.

[0503] In one embodiment of the present invention, the viral vector is a retroviral vector. The vector production system contains the Gag and Gag / Pol proteins and the Env protein. or nucleic acid sequences encoding these functional substitutes, and vectors containing the nucleic acid sequences of the present invention. The production system further comprises a nucleic acid sequence encoding a Rev protein and / or a genomic sequence. Alternatively, it may comprise a nucleic acid sequence encoding TRAP.

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

[0505] In another embodiment, the viral vector is derived from a lentivirus. ,Viral vectors include HIV-1, HIV-2, SIV, FIV, BIV, EIAV, It is derived from CAEV or Visna lentivirus.

[0506] Another aspect of the present invention is a method for increasing viral vector titers in eukaryotic vector-producing cells. a method for inducing the production of a viral vector of the present invention in a eukaryotic vector-producing cell, and introducing a nucleic acid sequence encoding TRAP into the system, wherein TRAP is a TRAP-binding and bind to the site or part of the NOI, thereby inhibiting translation of the NOI, thereby inhibiting the TRAP binding site. EP 1 237 363 A1 2 5 10 15 20 25 30 35 40 45 50 55 Method for increasing viral vector titer compared to viral vector without .

[0507] Another aspect of the present invention is a DNA vector for use in the viral vector production system of the present invention. Such DNA constructs (e.g., plasmids) contain the nucleic acid sequences of the present invention. The vector genome construct may comprise:

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

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

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

[0511] In one embodiment of the present invention, the set of DNA constructs encodes the Rev protein or a functional representative thereof. Further included are DNA constructs encoding the alternatives.

[0512] In one embodiment, the viral vector production system comprises a modular nucleic acid construct (modular Modular constructs are used in the production of lentiviral vectors. A modular construct is a DNA expression construct that contains two or more nucleic acids. The present invention provides a method for producing two or more nucleic acids for use in the production of lentiviral vectors. The nucleic acid may be a DNA plasmid containing the nucleic acid. The plasmid may be a bacterial plasmid. For example, it can encode gag-pol, rev, env, and a vector genome. Additionally, a modular system designed for packaging and producer cell line generation is also available. The constructs may contain transcriptional regulatory proteins (e.g., TetR, CymR) and / or translational repression proteins. Proteins (e.g., TRAP) and selectable markers (e.g., Zeocin ( (target), hygromycin, blasticidin, puromycin, neomycin resistance genes It may be necessary to further code the appropriate modules for use in the present invention. A modular construction is described in European Patent No. 3502260, which is incorporated by reference in its entirety. and is incorporated herein by reference.

[0513] Modular constructs for use in accordance with the present invention may contain retroviruses on one construct. It contains nucleic acid sequences encoding two or more of the gene components, so that these modules The safety profile of the modular construct is being studied and additional safety features are being incorporated into the construct. These features include multiple open-chain modifications of the retroviral vector components. Use of insulators and / or modular construction of reading frames These features include the specific orientation and arrangement of retroviral genes in the host. This prevents direct read-through to generate replication-competent viral particles. It is thought that this will be the case.

[0514] The nucleic acid sequences encoding the viral vector components are reversed and / or reversed in the modular construct. or alternate transcriptional orientation. Thus, the nucleic acid sequences encoding the viral vector components are not presented in the same 5' to 3' orientation, resulting in viral vector components being presented in the same mRNA. The reverse orientation means that the vector components cannot be produced from the NA molecule. At least two coding sequences are involved in "head-to-head" and "tail-to-tail" translation. This may mean that the sequences are presented in a transcription orientation, such as one on one strand of the modular construct. vector components, e.g., the coding sequence for env and another vector component on the opposite strand. This can be achieved by providing a coding sequence for the gene, e.g., rev. If coding sequences for more than two vector components are present in a modular construct, In this case, at least two of the coding sequences are present in opposite transcriptional orientations. When the coding sequences for the vector components of are present in a modular construct, each component is Any flanking coding sequence(s) to other vector components to which it flanks The coding sequences can be oriented such that they are in the opposite 5' to 3' orientation relative to each other, i.e., Alternating 5' to 3' (or transcription) orientations for the rows can be used.

[0515] Modular constructs for use in accordance with the present invention comprise the following vector components: gag- Encodes two or more of pol, rev, env, and the vector genome Modular constructs may contain nucleic acid sequences encoding any combination of vector components. In one embodiment, the modular construct may comprise a nucleic acid sequence comprising: 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 a functional substitute thereof; and Yobi 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 cells for producing the retroviral vector are selected from the group consisting of the cells i) to x) above. The nucleic acid sequences may include nucleic acid sequences encoding any one of a combination of The same locus can be located at a single chromosome within a cell, and can be in reverse and / or alternate orientations. extrachromosomal loci, e.g., a single plasmid, or a single locus within the genome of a cell ( A cell can be transfected with a retroviral vector, e.g., a lentiviral vector. The cells may be stable or transient for producing viral vectors.

[0517] Another aspect of the invention is the use of nucleic acid sequences, viral vector production systems or DNA constructs of the invention. The present invention relates to a viral vector-producing cell containing part or all of the above.

[0518] "Viral vector-producing cells" are cells that produce viral vectors or viral vector particles. Viral vector-producing cells are to be understood as cells capable of producing the viral vector. The viral vector system may be a "producer cell" or a "packaging cell." One or more DNA constructs are stably integrated into viral vector-producing cells. Alternatively, all viral vector systems can be expressed in a single vector or maintained episomally. The DNA components can be transiently transfected into viral vector-producing cells. Alternatively, producer cells stably expressing some of the components are transiently transfected with the remaining components. can be inserted.

[0519] The DNA expression cassette encoding TRAP was stably integrated into viral vector-producing cells. Alternatively, the TRAP-encoding D The NA expression cassette can be transiently transfected into viral vector-producing cells.

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

[0521] The level of repression required may vary depending on the NOI, so it is important to determine the level of repression required in the producing cells. The level of TRAPs achieved may also depend on the NOI. A combination of stable and transient TRAP expression may be desirable. Stable expression is While transient expression can provide continuous levels of TRAP expression in cells, transient expression can provide shorter durations of expression. This may provide for increased levels of TRAP expression over time, e.g., leading to more problematic / toxic For transgene suppression, a TRAP gene already present at the time of vector production (e.g., provided by stable expression) is used. Both ATP (provided) and high levels of TRAP may be beneficial.

[0522] Thus, in another embodiment of the invention, the production cell stably expresses a TRAP construct, The TRAP construct is also transiently expressed. Transient expression is similar to that provided by stable expression. This can provide higher levels of TRAP expression for a short period of time.

[0523] "Stable expression" refers to the long-term expression of TRAP from a construct that confers stable expression. It should be understood that this does not substantially change the

[0524] "Transient expression" refers to the expression of TRAP from a construct that confers transient expression over a long period of time. It should be understood that the vector is not stable. Preferably, the vector encodes a TRAP that confers transient expression. The polynucleotide is not integrated into the producer cell genome but is episomal in the producer cell. is not maintained.

[0525] As used herein, the term "packaging cells" refers to cells that contain infectious vector particles. Refers to cells that contain the elements necessary for the production of offspring but lack the vector genome. Such packaging cells express the viral structural proteins (gag, gag / p one or more expression cassettes capable of expressing the nucleotide sequences ol and env Contains:

[0526] The producer / packaging cells can be of any suitable cell type. The host cell is generally a mammalian cell, but can be, for example, an insect cell.

[0527] As used herein, a "producer / producing cell" or "vector producing cell" refers to a The term "vector-producing cells" refers to cells that are used for the production of retroviral vector particles and TRAP It refers to a cell that contains all the elements necessary for expression of

[0528] Producer cells can be stable producer cell lines or transiently induced producer cells. The antibody may be any of the following cell lines:

[0529] In one embodiment of the present invention, the envelope and nucleocapsid, TRAP, and rev nucleotide sequences, if present in the producer cell and / or However, any one of these sequences may be stably integrated into the packaging cells. Alternatively, more may be present in episomal form, with gene expression being regulated from the episome. The vector may be generated from a recombinant vector or may be transiently transfected into a production cell.

[0530] Vector-producing cells can be cells cultured in vitro, such as tissue culture cell lines. Suitable cell lines include mammalian cells such as mouse fibroblast-derived cell lines or human cell lines. Preferably, the vector-producing cells are derived from a human cell line. do.

[0531] In one aspect, the vector of the present invention comprises, as its production system, a gene operably linked to an NOI. The vector genome, gag-pol components, envelope and T Four transcription units are used to express RAP. The envelope expression cassette is VSV-G The ribosomal protein may contain one of a number of heterologous envelopes, such as a ribosomal protein, ...

[0532] Viral vector production process Another aspect of the invention is the use of nucleic acid sequences, viral vector production systems or DNA constructs of the invention. into a viral vector-producing cell; and and culturing the producer cells under conditions suitable for the production of the viral vector. This relates to a method for

[0533] Suitable "producer cells" are those that can produce viral vectors or viruses when cultured under appropriate conditions. Suitable "producer cells" are generally cells capable of producing vector particles. Mammalian or human cells, such as HEK293T, HEK293, CAP, CAP-T or CHO cells, but may also be insect cells such as SF9 cells.

[0534] The producer cells can also be avian cells, such as EB66® (Sigma) cells. Avian cells have been used in the production of virus-based vaccines in human and veterinary medicine, e.g., influenza and and may be particularly useful for the production of Newcastle disease virus vaccines.

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

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

[0537] Another aspect of the present invention is to produce the viral vector of the present invention by the viral vector production system of the present invention. Viral vectors produced using vector-producing cells or by the methods of the present invention Regarding.

[0538] In one embodiment, the viral vector particle comprises a nucleic acid sequence of the invention. The particle may be derived from a retrovirus, an adenovirus, or an adeno-associated virus. Lentiviral vector particles can be derived from lentiviruses. The child is infected with HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or It may be derived from a snailentivirus.

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

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

[0541] "Cells transduced by viral vector particles" refers to cells transduced by viral vector particles. The term "cell" should be understood as referring to the cells into which the nucleic acid delivered by the vector is introduced, in particular the target cells. .

[0542] use Another aspect of the invention is the use of a viral vector of the invention or a method of treating atopic dermatitis with a viral vector of the invention for use in medicine. The present invention relates to cells or tissues transduced with the viral vectors of the invention.

[0543] Another aspect of the present invention is the use of a viral vector of the present invention or a virus of the present invention in medicine. Uses of cells or tissues transduced with the vector.

[0544] Another aspect of the invention is to provide a method for inserting a nucleotide of interest into a target site requiring the nucleotide of interest. The viral vector of the present invention for preparing a medicament for delivering a methicone Use of producer cells or cells or tissues transduced with the viral vectors of the invention do.

[0545] Such uses of the viral vectors or transduced cells of the invention are described herein. As described, this may be for therapeutic or diagnostic purposes.

[0546] Therapeutic Vectors Retroviral Therapeutic Vectors In one embodiment, the retroviral vector of the present invention is used to treat Parkinson's disease. was used to introduce three genes encoding three enzymes of the dopamine synthesis pathway into the Retroviral vectors can be expressed as VSV-G or alternative viral envelope proteins. Human immunodeficiency virus (HIV) or equine infectious anemia virus (EVI) that can be pseudotyped with HIV. It is a non-replicating, self-inactivating minimal lentiviral vector derived from EIAV. The gene carried by the viral vector is a truncated form of 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 The three enzymes can be encoded by three separate open reading frames. It may be encoded in frame by a retroviral vector. The vector contains the TH and CH1 enzymes in the first open reading frame and the The gene may encode a fusion with the AADC enzyme in the open reading frame. The expression may be driven by a CMV promoter and the expression cassette may contain one or more The retroviral vector may contain an IRES element. The retroviral vector may be injected directly into the striatum of the brain. It can be administered by

[0547] In another embodiment, the retroviral vector of the present invention is a vector that expresses photoreceptor cells and their supporting network. The MYO7A gene for correction was introduced into RPE cells, thereby Gene therapy designed to attenuate or reverse vision loss associated with Shah 1B syndrome The retroviral vector may be VSV-G or an alternative virus. Human immunodeficiency virus (HIV) or equine viral load can be pseudotyped with envelope proteins A non-replicating, self-inactivating minimal lentiviral vector derived from infectious anemia virus(EIAV). The gene carried by the retroviral vector encodes the MYO7A protein. The MYO7A cDNA (a large gene over 100 mb in length) encodes The expression of the MYO7A gene was controlled by the CMV promoter and the CMV / MYO7A chimeric promoter. Retroviral vectors can be driven by a promoter other than the α- or α-terminal promoter. It can be administered by direct subretinal injection after vitrectomy.

[0548] In another embodiment, the retroviral vector of the present invention contains an ATP-binding cassette for correction. The ABCA4 gene (also known as ABCR) was introduced into photoreceptor cells, thereby It may be used to attenuate or reverse the pathophysiology that leads to Targardt's disease. Viral vectors pseudotyped with VSV-G or alternative viral envelope proteins Human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV) may cause It is a non-replicating, self-inactivating minimal lentiviral vector derived from a retroviral vector. The gene carried by the ABCA4 gene is the ABCA4 cDNA that encodes the ABCA4 protein. The expression of the ABCA4 gene is controlled by the CMV promoter, photoreceptor-specific promoters, e.g. For example, it may be driven by rhodopsin kinase or an alternative promoter. The vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0549] In another embodiment, the retroviral vector of the present invention is used to treat wet age-related macular degeneration (AMD). ), abnormal blood vessel growth and development in the eyes of patients with diabetic macular edema or retinal vein occlusion and / or to prevent the recurrence of vascular leakage, and / or to treat dry age-related macular degeneration ( A gene therapy designed to prevent abnormal blood vessel growth in the eyes of patients with AMD This retroviral vector can be used as a therapeutic product. or one or more genes encoding one or more anti-angiogenic proteins, such as endostatin Retroviral vectors deliver genes or multiple genes. Human immunodeficiency virus (HIV) or viral envelope proteins that can be pseudotyped with A non-replicating, self-inactivating minimal lentiviral vector derived from infectious anemia virus (EIAV) In one embodiment, the retroviral vector is a vector for delivery to retinal pigment epithelial cells. For this purpose, we used an internal ribosome entry site (IRES) to construct a bicistronic human Anti-angiogenic (one or more) cells expressing the angiostatin and angiostatin genes Expression of genes is controlled by CMV, RPE-specific promoters, e.g., vitelloid macular dystrophy The VMD2 promoter (more recently known as the bestrophin promoter) It can be driven by the promoter of the retroviral vector (as described above) or by an alternative promoter. The tar can be administered by direct subretinal injection after vitrectomy of the eye.

[0550] In another embodiment, the retroviral vector of the present invention is used to treat wet age-related macular degeneration (AMD). ) Gene therapy designed to prevent recurrence of abnormal blood vessel growth in patients' eye edema This retroviral vector can be used as a product. or endostatin, encoding one or more anti-angiogenic proteins. Retroviral vectors deliver multiple genes. derived from equine infectious anemia virus (EIAV) that can be pseudotyped with the HIV envelope protein In one embodiment, the vector is a non-replicating, self-inactivating minimal lentiviral vector. The viral vector contains an internal ribosome entry site (IRS) for delivery to retinal pigment epithelial cells. Using the bicistronic RES (Regenerated Endostatin and Angiostatin Genes) Expression of one or more anti-angiogenic genes is CMV-RPE-specific. Promoters, such as the vitelloid macular dystrophy 2 (VMD2) promoter (more recently known in the literature as the bestrophin promoter) or by alternative promoters. Retroviral vectors can be delivered directly subretinally after vitrectomy of the eye. It may be administered by injection.

[0551] In another embodiment, the retroviral vector of the present invention is delivered to the donor cornea prior to transplantation (1 Corneal graft rejection as a result of angiogenesis due to delivery of anti-angiogenic genes (one or more) Retroviral vectors can be used as gene therapy products designed to prevent infection. can be pseudotyped with VSV-G, Ebola or alternative viral envelope proteins Human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV) In one embodiment, the retroviral vector is a non-replicating, self-inactivating minimal lentiviral vector. The virus vector contains an internal ribosome entry site ( Bicistronic human endostatin and angiostatin using IRES A retroviral vector expressing an anti-angiogenic gene(s), such as a gene. The transduced donor tissue can also be preserved prior to transplantation. The expression of the anti-angiogenic gene(s) can be driven by a promoter such as a CMV promoter. However, alternative promoters may be used. It is also possible that this can be done.

[0552] In another embodiment, the retroviral vector of the present invention is used to treat wet age-related macular degeneration (AMD). ), abnormal blood vessel growth and development in the eyes of patients with diabetic macular edema or retinal vein occlusion and / or to prevent the recurrence of vascular leakage, and / or to treat dry age-related macular degeneration ( A gene therapy designed to prevent abnormal blood vessel growth in the eyes of patients with AMD This retroviral vector expresses a soluble form of the fms-like tyrosine kinase inhibitor, which can be used as a therapeutic agent. Retroviral vector delivers the gene encoding the kinase (soluble Flt-1) human immune cells that can be pseudotyped with VSV-G or alternative viral envelope proteins non-replicating viruses derived from HIV or equine infectious anemia virus (EIAV) This is a self-inactivating minimal lentiviral vector. Expression of soluble Flt-1 gene is achieved by CM. V, RPE-specific promoter, e.g., vitelloid macular dystrophy 2 (VMD2) promoter promoter (more recently known as the bestrophin promoter), and The retroviral vector can be driven by an alternative promoter. It can be administered by direct subretinal injection after removal.

[0553] In another embodiment, the retroviral vector of the present invention is used to treat wet age-related macular degeneration (AMD). ), abnormal blood vessel growth and development in the eyes of patients with diabetic macular edema or retinal vein occlusion and / or to prevent the recurrence of vascular leakage, and / or to treat dry age-related macular degeneration ( A gene therapy designed to prevent abnormal blood vessel growth in the eyes of patients with AMD This retroviral vector can be used as a therapeutic product. Retroviral vectors deliver one or more genes encoding protein (PEDF). is a human immunoglobulin that can be pseudotyped with VSV-G or alternative viral envelope proteins. Non-replicating viruses derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV) It is a self-inactivating minimal lentiviral vector. PEDF gene expression is mediated by CMV, oocyte The macular dystrophy 2 (VMD2) promoter (more recently the bestrophin promoter) by RPE-specific promoters such as the ER promoters (known as ER promoters) or alternative promoters The retroviral vector can be driven by a promoter. It may be administered by subretinal injection.

[0554] In another embodiment, the retroviral vector of the present invention is used to treat wet age-related macular degeneration (AMD). ), abnormal blood vessel growth and development in the eyes of patients with diabetic macular edema or retinal vein occlusion and / or to prevent the recurrence of vascular leakage, and / or to treat dry age-related macular degeneration ( A gene therapy designed to prevent abnormal blood vessel growth in the eyes of patients with AMD This retroviral vector can be used as a therapeutic product. F) inhibitors, such as anti-VEGF antibodies or binding fragments thereof (e.g., aflibercept) , including but not limited to, VEGF-specific aptamers, or soluble forms of VEGF receptors. VEGF-blocking peptides or polypeptides and / or platelet-derived growth factors not inhibitors of PDGF, such as anti-PDGF antibodies or binding fragments thereof, PDGF-specific antibodies, PDGF receptor antagonists, including but not limited to, PDGF receptor agonists, PDGF receptor antagonists, or soluble forms of the PDGF receptor. delivery of a gene or genes encoding a truncated peptide or polypeptide Retroviral vectors can be expressed using VSV-G or alternative viral envelope proteins. Human immunodeficiency virus (HIV) or equine infectious anemia virus (E) can be pseudotyped in In one embodiment, the vector is a non-replicating, self-inactivating minimal lentiviral vector derived from IAV (IaV). In this study, retroviral vectors were used to target intracellular ribosomes for delivery to retinal pigment epithelial cells. Inhibitors of VEGF and PDGF in a bicistronic configuration using an IRES (insertion-restricted expression system) The expression of the gene(s) is associated with CMV, vitelloid macular dystonia, and The trophin 2 (VMD2) promoter (more recently known as the bestrophin promoter) These promoters are either RPE-specific promoters, such as the known ribosomal promoters, or alternative promoters. Retroviral vectors can be delivered by direct subretinal injection after vitrectomy of the eye. It can be administered accordingly.

[0555] In another embodiment, the retroviral vector of the present invention is a vector encoding a corrective gene for vitelloid macular dysplasia. MicroRNAs specific for VMD2 and disease-associated forms of VMD2 A cassette encoding a miRNA or a corrected RDS gene encoding peripherin 2 Genes and cassettes encoding miRNAs specific for disease-associated forms of RDS are identified. and Best's disease or Best's vitelliform macular degeneration (BVMD). Retroviral vectors can be used to attenuate or reverse the pathophysiology that leads to The target can be pseudotyped with VSV-G or alternative viral envelope proteins. Non-complex viruses derived from the immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV) It is a self-inactivating minimal lentiviral vector manufactured by . Gene expression is controlled by CMV, egg yolk-like yolk vector. The macula dystrophy 2 (VMD2) promoter (and more recently the bestrophin promoter) These promoters are either RPE-specific promoters, such as the α-glucanase (known as α-glucanase), or alternative promoters. Retroviral vectors can be delivered directly to the retina after vitrectomy of the eye. It may be administered by subcutaneous injection.

[0556] In another embodiment, the retroviral vector of the present invention comprises a corrected retinaldehyde binding tag. The RLBP1 gene, RLBP1, was introduced into retinal pigment epithelial cells, thereby It may be used to attenuate or reverse the pathophysiology that leads to retinal dystrophies. Retroviral vectors are expressed with VSV-G or alternative viral envelope proteins. Can be pseudotyped with human immunodeficiency virus (HIV) or equine infectious anemia virus (EIA) It is a non-replicating, self-inactivating minimal lentiviral vector derived from retrovirus V. The gene carried by the vector is RLBP1cD, which encodes the RLBP1 protein. RLBP1 gene expression is associated with CMV, vitelloid macular dystrophy 2 (VMD), and 2) promoters (more recently known as the bestrophin promoter), etc. can be driven by an RPE-specific promoter or by an alternative promoter Retroviral vectors can be administered by direct subretinal injection after vitrectomy of the eye. .

[0557] In another embodiment, the retroviral vector of the present invention is designed to treat glaucoma. This retroviral vector can be used as a gene therapy product to reduce intraocular pressure. COX-2 and / or prostaglandin F2α receptors (F Retroviral vectors deliver one or more genes encoding the VPR. Human immunodeficiency virus (HIV) that can be pseudotyped with SV-G or alternative viral envelope proteins non-replicating self-infecting viruses derived from HIV or equine infectious anemia virus (EIAV) In one embodiment, the retroviral vector is a live, minimal lentiviral vector. Bisectin using an internal ribosome entry site (IRES) for delivery to the anterior chamber of the eye The genes expressing the COX-2 and prostaglandin F2α receptor (FPR) genes in the chromosome 111 are Expression of the gene(s) is driven by a CMV or alternative promoter. The retroviral vector can be administered by transcorneal injection.

[0558] In another embodiment, the retroviral vector of the present invention introduces a corrected harmonin gene. and used to attenuate or reverse the pathophysiology that leads to Usher syndrome 1c Retroviral vectors can be expressed as VSV-G or alternative viral envelope proteins. Human immunodeficiency virus (HIV) or equine infectious anemia virus (EVI) that can be pseudotyped with HIV. It is a non-replicating, self-inactivating minimal lentiviral vector derived from EIAV. The gene carried by the virus vector is the harmonin gene encoding the harmonin protein. The harmonin gene is expressed under the control of the CMV promoter or an alternative promoter. Retroviral vectors can be delivered directly to the retina after vitrectomy of the eye. It may be administered by subcutaneous injection.

[0559] In another embodiment, the retroviral vector of the present invention comprises a corrective Rab escort protein. Transfection of the REP1 gene to attenuate or reverse the pathophysiology leading to total choroidal atrophy Retroviral vectors can be used to transduce VSV-G or alternative viruses. Human immunodeficiency virus (HIV) or viral envelope proteins that can be pseudotyped with A non-replicating, self-inactivating minimal lentiviral vector derived from infectious anemia virus (EIAV) The gene carried by the retroviral vector is the REP1 protein. The REP1 cDNA encodes the REP1 gene. The retroviral vector may be driven by a promoter other than the promoter of the vitreous of the eye. It may be administered by direct subretinal injection after excision.

[0560] In another embodiment, the retroviral vector of the present invention comprises a correction loop(s). Nucleotide-gated channel beta 2 (CNGB2) and / or cyclic nucleotides The sensitive channel alpha 3 (CNGA3) gene was introduced into the eye to cause color blindness. Retroviral vectors can be used to attenuate or reverse the pathophysiology of V Human immunodeficiency virus (HIV) that can be pseudotyped with SV-G or alternative viral envelope proteins non-replicating self-infecting viruses derived from HIV or equine infectious anemia virus (EIAV) It is a minimal active lentiviral vector. It is carried by a retroviral vector (1 The gene or genes encode the CNGB2 and / or CNGA3 proteins. The CNGB2 and / or CNGA3 genes (one or more) are involved in the Expression of multiple (or multiple) genes is driven by the CMV promoter or an alternative promoter The retroviral vector can be administered by direct subretinal injection after vitrectomy of the eye. It can be done.

[0561] In another embodiment, the retroviral vector of the present invention delivers a corrected CEP290 gene to the eye. to attenuate or reverse the pathophysiology leading to Leber congenital amaurosis (LCA). Retroviral vectors can be used to induce the expression of VSV-G or alternative viruses. Human immunodeficiency virus (HIV) or equine viral load can be pseudotyped with envelope proteins A non-replicating, self-inactivating minimal lentiviral vector derived from infectious anemia virus(EIAV). The gene carried by the retroviral vector is located in the 290 kDa centrosomal tag. The CEP290 gene encodes a protein. Expression of the CEP290 gene is The retroviral vector may be driven by a promoter or an alternative promoter. can be administered by direct subretinal injection after vitrectomy of the eye.

[0562] In another embodiment, the retroviral vector of the present invention is a corrective retinitis pigmentosa GTP-associated The RPGR gene was introduced into the eye to treat X-linked retinitis pigmentosa. Retroviral vectors can be used to attenuate or reverse the pathophysiology that results from , human immunodeficiency virus (HIV) that can be pseudotyped with VSV-G or alternative viral envelope proteins Non-replicating autoantibodies derived from whole virus (HIV) or equine infectious anemia virus (EIAV) It is a self-inactivating minimal lentiviral vector. It is carried by a retroviral vector. The gene is the RPGR cDNA that encodes the RPGR protein. Expression of the retrovirus can be driven by the CMV promoter or an alternative promoter. The viral vector can be administered by direct subretinal injection after vitrectomy of the eye.

[0563] In another embodiment, the retroviral vector of the present invention comprises a corrected retinoschisin 1 (RS 1) Introducing a gene into the eye to cause X-linked retinochisis Retroviral vectors can be used to attenuate or reverse the pathophysiology of Human immunodeficiency viruses that can be pseudotyped with VSV-G or alternative viral envelope proteins Non-replicating self derived from viruses (HIV) or equine infectious anemia virus (EIAV) It is an inactive minimal lentiviral vector. The gene is RS1 cDNA, which encodes the RS1 protein. Expression of the RS1 gene is It can be driven by the CMV promoter or an alternative promoter. The drug can be administered by direct subretinal injection after vitrectomy of the eye.

[0564] In another embodiment, the retroviral vector of the present invention is a vector for the treatment of retinitis pigmentosa 1 (RP1). 1) Introducing genes into the eye to attenuate or reverse the pathophysiology that leads to retinitis pigmentosa Retroviral vectors can be used to induce the expression of VSV-G or alternative viruses. Human immunodeficiency virus (HIV) or equine viral load can be pseudotyped with envelope proteins A non-replicating, self-inactivating minimal lentiviral vector derived from infectious anemia virus(EIAV). The gene carried by the retroviral vector encodes the RP1 protein. The RP1 gene is expressed by the CMV promoter and the photoreceptor cell-specific driven by a specific promoter, e.g., rhodopsin kinase or an alternative promoter The retroviral vector is administered by direct subretinal injection after vitrectomy of the eye. obtain.

[0565] In another embodiment, the retroviral vector of the present invention comprises a retinal pigment epithelium-specific 65 Leber congenital amaurosis type 2 (LCA) was treated by introducing the 200 kDa protein (RPE65) gene. Retroviral vectors can be used to attenuate or reverse the pathophysiology that leads to is a human immunoglobulin that can be pseudotyped with VSV-G or alternative viral envelope proteins. Non-replicating viruses derived from human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV) It is a self-inactivating minimal lentiviral vector. The gene is RPE65 cDNA, which encodes the RPE65 protein. The expression of the 5 genes is driven by the CMV vitelloid macular dystrophy 2 (VMD2) promoter ( RPE-specific promoters, such as the bestrophin promoter (more recently known as the The retroviral vector may be driven by the promoter or by an alternative promoter. The tar can be administered by direct subretinal injection after vitrectomy of the eye.

[0566] In another embodiment, the retroviral vector of the present invention comprises a corrected human proline / arginine The PRELP gene was introduced to inhibit exudation. Age-related macular degeneration (AMD), dry AMD, diabetic macular edema, or retinal vein occlusion Retroviral vectors can be used to attenuate or reverse the pathophysiology of Human immunodeficiency viruses that can be pseudotyped with VSV-G or alternative viral envelope proteins Non-replicating self derived from viruses (HIV) or equine infectious anemia virus (EIAV) It is an inactive minimal lentiviral vector. The gene is the PRELP cDNA that encodes the PRELP protein. Expression of the CMV vitelloid macular dystrophy 2 (VMD2) promoter (more recently RPE-specific promoters such as the bestrophin promoter (known in Retroviral vectors can be driven by the promoters listed above or by alternative promoters. , can be administered by direct subretinal injection after vitrectomy of the eye.

[0567] In another embodiment, the retroviral vector of the invention comprises a synthetic myocilin-specific miR. By introducing a nucleic acid sequence encoding NA into the eye, the expression of myocilin is knocked down. Used to attenuate or reverse the pathophysiology that leads to juvenile open-angle glaucoma Retroviral vectors can be expressed as VSV-G or alternative viral envelope tags. Human immunodeficiency virus (HIV) or equine infectious anemia virus can be pseudotyped with HIV proteins. It is a non-replicating, self-inactivating minimal lentiviral vector derived from Epstein-Barr virus (EIAV). Myocilin-specific miRNA expression is driven by the CMV promoter or alternative promoters. Retroviral vectors can be delivered by direct subretinal injection after vitrectomy of the eye. It can be administered accordingly.

[0568] In another embodiment, the retroviral vector of the present invention encodes a nucleotide sequence encoding a nucleotide from the glutathione biosynthetic pathway. The rate-limiting enzyme(s) glutamate-cysteine ​​ligase (GCL) and and / or glutathione synthetase (GSS), and / or synthetic gamma-glutamate A nucleic acid sequence encoding a specific miRNA for GGT was introduced into the eye. and gene augmentation and / or knockdown to induce retinitis pigmentosa. Retroviral vectors can be used to attenuate or reverse pathophysiology, e.g. , human immunodeficiency virus (HIV) that can be pseudotyped with VSV-G or alternative viral envelope proteins Non-replicating viruses derived from whole viruses (HIV) or equine infectious anemia virus (EIAV) A self-inactivating minimal lentiviral vector. Expression of the GSS gene and / or synthetic GGT-specific miRNA is associated with CMV proliferation. It may be driven by a motor or an alternative promoter. Vectors utilize one or more internal ribosome entry sites (IRES) to deliver the vector to the host. Expresses 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 vector of the present invention is used to treat frontotemporal dementia, Alzheimer's disease, and other conditions. Neurodegenerative disorders such as Immersion disease, Parkinson's disease, and Huntington's disease, and amyotrophic lateral sclerosis gene therapy products designed to treat motor neuron disorders such as amyotrophic lateral sclerosis (ALS) and This retroviral vector can be used to express VEGF. 145 , VEGF 165 too Or VEGF 189 can be VEGF-A isoforms such as VEGF-B a gene encoding a VEGF protein, which may be VEGF-C or VEGF-D Retroviral vectors deliver genes that have neuroprotective effects. can be pseudotyped with VSV-G or VSV-G or alternative viral envelope proteins. non-human avian immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV) It is a replicating, self-inactivating minimal lentiviral vector. Gene expression is mediated by CMV or Retroviral vectors can be driven by alternative promoters. or by direct injection into the cerebrospinal fluid by intrathecal or intraventricular injection It can be administered accordingly.

[0570] In another embodiment, the retroviral vector of the present invention is adapted to treat cystic fibrosis. This retroviral vector can be used as a gene therapy product specifically designed for cysts. delivers the gene encoding the fibrosis transmembrane conductance regulator (CFTR). The viral vector also contains influenza hemagglutinin and Sendai virus envelope F. or HN, Ebola, baculovirus GP64 or alternative viral envelope proteins Human immunodeficiency virus (HIV) or equine infectious anemia virus that can be pseudotyped with proteins It is a non-replicating, self-inactivating minimal lentiviral vector derived from EIAV. Expression of the gene can be driven by a CMV or alternative promoter. The drug is administered intranasally, by use of a nebulizer, or into the lungs via bronchoalveolar lavage. The drug may be administered by direct delivery into the body.

[0571] In another embodiment, the retroviral vector of the invention comprises an orthogonal N-sulfoglucosamine Sulfohydrolase (SGSH) and / or sulfatase modifying factor 1 (SUMF1 ) gene(s) into the brain to induce the pathogenesis that leads to Sanfilippo syndrome A. Retroviral vectors can be used to attenuate or reverse the process of VSV-G. or human immunodeficiency virus (HIV) that can be pseudotyped with alternative viral envelope proteins ( Non-replicating, self-inactivating miniviruses derived from HIV or equine infectious anemia virus (EIAV) Lentiviral vectors. Retroviral vectors (one or more) The genes include SGSH cDNA and / or S The SUMF1 gene (or genes) encodes the UMF1 protein. Expression of the gene may be driven by a CMV promoter or an alternative promoter. In this study, retroviral vectors utilize an internal ribosome entry site (IRES) The bicistronic SGSH and SUMF1 genes can be expressed using the retrovirus. The vector may be administered by direct intracerebral injection.

[0572] In another embodiment, the retroviral vector of the present invention comprises an orthogonal acid alpha-glycosidase ( GAA) gene into large muscle groups and / or the lungs, resulting in Pompe disease This retroviral vector can be used to attenuate or reverse GA Retroviral vectors deliver genes encoding influenza A proteins. Hemagglutinin, Sendai virus envelope F or HN, Ebola, Baculovirus Pseudomonas aeruginosa with rabies G, VSV-G or alternative viral envelope proteins Typeable human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV) ) is a non-replicating, self-inactivating minimal lentiviral vector derived from Retroviral vectors can be driven by CMV, CMV, or alternative promoters. (i) direct injection into large muscle groups and / or (ii) intranasally using a nebulizer or by direct delivery into the lungs via bronchoalveolar lavage. .

[0573] In another embodiment, a nucleic acid encoding a CD19-specific chimeric antigen receptor (CAR19) The retroviral vectors of the invention can be used to transduce autologous or allogeneic T cells with the sequences These transduced T cells can then be used ex vivo to express CD19. Retroviral vectors are injected into subjects to treat cancers and leukemias. Human immunodeficiency viruses that can be pseudotyped with VSV-G or alternative viral envelope proteins Non-replicating self derived from viruses (HIV) or equine infectious anemia virus (EIAV) It is an inactive minimal lentiviral vector. Expression of the nucleic acid sequence encoding the CAR is mediated by EF It can be driven by 1α, CMV or alternative promoters.

[0574] In another embodiment, a nucleic acid sequence encoding a 5T4-specific chimeric antigen receptor (CAR) The retroviral vectors of the present invention are expressed in a manner that allows for the transduction of autologous or allogeneic T cells. These transduced T cells can then be used in vivo to treat cancers and tumors expressing 5T4. The retroviral vector is injected into a subject to treat VSV- Human immunodeficiency viruses that can be pseudotyped with G or alternative viral envelope proteins Non-replicating, self-inactivating viruses derived from HIV or equine infectious anemia virus (EIAV) It is a small lentiviral vector. Expression of the nucleic acid sequence encoding 5T4 CAR is achieved by EF It can be driven by 1α, CMV or alternative promoters.

[0575] As known to those skilled in the art, there are a variety of proteins specific for cancer or leukemia-associated polypeptides. Any cancer or leukemia-associated polypeptide can be produced using a CAR. A nucleic acid sequence encoding a chimeric antigen receptor (CAR) specific for a specific peptide, either autologous or homologous, is used. The retroviral vectors of the invention are used ex vivo to transduce seed T cells. These transduced T cells can then be used to treat the cancer or leukemia associated with the CAR. The retroviral vectors are injected into subjects to treat cancers and leukemias that express the retroviral polypeptide. Viral vectors pseudotyped with VSV-G or alternative viral envelope proteins Human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV) may cause It is a non-replicating, self-inactivating minimal lentiviral vector derived from a CAR-encoding nucleic acid. Expression of the nucleic acid sequence can be driven by EF1α, CMV, or alternative promoters. Suitable cancer- or leukemia-associated polypeptides that can be targeted by the CAR include: These include, but are not limited to: mesothelin, folate receptor alpha, immunoglobulin κ light chain, CD30, carcinoembryonic antigen (CEA), CD138, ganglioside G2 (GD2 ), CD33, CD22, epidermal growth factor receptor (EGFR) such as EGF-VIII, I L-13Rα2, CD20, ErbB such as Her2, prostate-specific membrane antigen (PSMA) , Lewis Y antigen and fibroblast activation protein (FAB).

[0576] In another embodiment, peptide-M expressed on diseased cells, leukemia cells or cancerous cells. Nucleic acid sequences encoding T cell receptors (TCRs) specific for HCs are used to identify autologous or allogeneic T The retroviral vectors of the present invention can be used ex vivo to transduce cells. Then, a disease, cancer or leukemia associated with the expression of peptide-MHC that binds the TCR is detected. These transfected T cells are infused into a subject to treat retroviruses. The vector can be pseudotyped with VSV-G or an alternative viral envelope protein. Human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV) The nucleic acid sequence encoding the TCR is a non-replicating, self-inactivating minimal lentiviral vector. Expression of the present invention can be driven by EF1α, CMV or an alternative promoter. The TCR encoded by the vector may be a single-chain TCR (scTCR) or a dimeric TCR. As known to those skilled in the art, suitable dTCRs include those derived from the International Suitable scTCRs include those described in Patent Publication No. 2003 / 020763. Examples of such compounds include those described in International Publication No. 1999 / 018129. In certain aspects of the present invention, the present invention treats AIDS, leukemia, and cancers, including myeloma and sarcoma. To do this, T cells transfected with a TCR can be used.

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

[0578] In another embodiment, the retroviral vector of the present invention encodes adenosine deaminase. It is used to treat severe combined immunodeficiency syndrome (ADA) by introducing a gene encoding the Retroviral vectors may be used containing VSV-G or alternative viral envelopes. Human immunodeficiency virus (HIV) or equine infectious anemia virus (EIV) can be pseudotyped with the protein A non-replicating, self-inactivating minimal lentiviral vector derived from HIV-1 (EIAV) Gene expression is 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 used in the treatment of diseases. The compound can be injected into a subject.

[0579] In another embodiment, the retroviral vector of the invention encodes a WAS protein. Used to treat Wiskott-Aldrich syndrome (WAS) by introducing a gene Retroviral vectors can be expressed as VSV-G or alternative viral envelope tags. Human immunodeficiency virus (HIV) or equine infectious anemia virus can be pseudotyped with HIV proteins. A non-replicating, self-inactivating minimal lentiviral vector derived from EIAV (E1V-1) Gene expression is driven by the CMV promoter or an alternative promoter. The retroviral vectors of the present invention are used ex vivo to transduce bone marrow stem cells. These transduced bone marrow stem cells can then be used to treat disease. It can be injected into a subject.

[0580] In another embodiment, the retroviral vector of the present invention comprises a wild-type β-globin, a wild-type One of several globins, including fetal globin and mutated "anti-sickling" globin used to treat sickle cell disease or thalassemia by introducing a gene encoding one of As known to those skilled in the art, examples of anti-sickling globins include those described in WO 2014 / 013494 and WO 2014 / 013494. These include those described in International Publication No. 043131 and International Publication No. WO 1996 / 009385. Retroviral vectors include, but are not limited to, VSV-G or alternative viral vectors. Human immunodeficiency virus (HIV) or A non-replicating, self-inactivating minimal lentivirus derived from equine infectious anemia virus (EIAV) Gene expression is driven by the CMV promoter or an alternative promoter. The retroviral vectors of the present invention can be used to transduce bone marrow stem cells. These transduced bone marrow stem cells can then be used ex vivo to treat disease. The device can be injected into a subject to place the device.

[0581] In another embodiment, the retroviral vector of the invention contains a corrective gene, Factor VIII. can be introduced into liver, muscle, or fat cells and used to treat hemophilia A. Viral vectors pseudotyped with VSV-G or alternative viral envelope proteins Human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV) may cause It is a non-replicating, self-inactivating minimal lentiviral vector derived from retroviruses. The gene carried by the vector is factor VIII. Expression can be driven by the CMV promoter or an alternative promoter.

[0582] In another embodiment, the retroviral vector of the present invention expresses a corrective gene, factor IX, in the liver. It can be introduced into liver, muscle, or fat cells and used to treat hemophilia B. The vectors are pseudotyped with VSV-G or alternative viral envelope proteins. derived from the human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV) It is a non-replicating, self-inactivating minimal lentiviral vector derived from a retrovirus. The gene carried by the vector is factor IX. Expression of the factor IX gene is It may be driven by the V promoter or an alternative promoter.

[0583] In another embodiment, the retroviral vector of the present invention encodes alpha-galactosidase A (α- GAL A) can be used to treat Fabry disease Retroviral vectors can be engineered to express VSV-G or alternative viral envelope proteins. Human immunodeficiency virus (HIV) or equine infectious anemia virus (EI) that can be pseudotyped with It is a non-replicating, self-inactivating minimal lentiviral vector derived from retrovirus (AV). The gene carried by the vector is GLA, which encodes the α-GAL A protein. The expression of the gene is driven by the CMV promoter or an alternative promoter. The retroviral vectors of the present invention can be driven by hematopoietic CD34 + Transducing stem cells These transduced hematopoietic CD3 4 + The stem cells can be injected into a subject to treat a disease.

[0584] In another embodiment, the retroviral vector of the present invention comprises a gene encoding a defective enzyme. It can be used to transfer genes to treat a form of porphyria. Vectors can be pseudotyped with VSV-G or alternative viral envelope proteins derived from the human immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV) It is a non-replicating, self-inactivating minimal lentiviral vector. The gene carried by the gene is selected from the table below for the type of porphyria to be treated. The gene encodes a defective enzyme related to the CMV promoter. or alternative promoters.

[0585] [Table 1]

[0586] In another embodiment, the retroviral vector of the present invention comprises a gene encoding a defective enzyme. It can be used to transfer genes to treat a form of mucopolysaccharidosis. The target can be pseudotyped with VSV-G or alternative viral envelope proteins. Non-complex viruses derived from the immunodeficiency virus (HIV) or equine infectious anemia virus (EIAV) It is a self-inactivating minimal lentiviral vector that can be easily produced. The gene to be delivered is a gene associated with the type of mucopolysaccharidosis to be treated selected from the table below. The gene is expressed by a CMV promoter or an alternative promoter. The promoter may be driven by:

[0587] [Table 2]

[0588] Construction of retroviral therapeutic vectors The retroviral vector of the present invention comprises four plasmids: (1) a nucleic acid encoding the required transgene(s) and nucleic acid sequence of the present invention; a recombinant retroviral vector genome plasmid for (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) The required transgene(s), the nucleic acid sequence of the invention, and the RRE a recombinant HIV vector genome plasmid encoding the 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, the retroviral vectors of the present invention, such as HIV, may be viral vectors and At least one modular construct encoding the components required for the production of TRAP The viral genome can be generated by transient transfection of HEK293T cells with the construct. , which contain the nucleic acid sequences of the present invention. Suitable modular constructs include those described in European Patent No. 3502260 These include, but are not limited to, those described in US Pat. No. 6,233,999.

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

[0592] Alternatively, the retroviral vector of the present invention may comprise (1) gag / pol, (2) env and (3) TRAP, and in the case of HIV vectors, a package that stably expresses Rev. The required transduction(s) can be produced by using aging cells. The recombinant retroviral vector genome encoding the transgene and the nucleic acid sequence of the present invention is The loading plasmid contains the RRE sequence in the case of an HIV vector, and is transfected by transient transfection. is introduced into such cells.

[0593] Alternatively, the retroviral vector of the present invention may comprise (1) gag / pol, (2) env (3) TRAP; (4) the required transgene(s) and the gene of the present invention. Producer cells stably expressing recombinant EIAV vector genome encoding nucleic acid sequences It can be produced in

[0594] Alternatively, the HIV vector of the present invention may comprise: (1) gag / pol; (2) env; (3) TRAP, (4) the required transgene(s), the nucleic acid sequence of the present invention, and (5) a recombinant HIV vector genome encoding the REV and RRE sequences; It can be produced in an expressing producer cell.

[0595] AAV therapeutic vectors In another embodiment, the AAV vectors of the invention are used in vivo to treat Parkinson's disease. It can be used to introduce three genes encoding three enzymes of the phosphodiesterase synthesis pathway. The gene carried by the AAV vector is a truncated form of human tyrosine hydroxylase (T H*) 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 The three enzymes can be encoded by three separate open reading frames. Alternatively, the AAV vector may be encoded by a first The TH and CH1 enzymes in the open reading frame and the second open reading frame The gene may encode a fusion with the AADC enzyme in frame. Expression of the gene is controlled by a CMV promoter. The expression cassette may be driven by one or more IRES elements. The AAV vector may be administered by direct injection into the striatum of the brain.

[0596] In another embodiment, the AAV vectors of the present invention are expressed on photoreceptor cells and the retinal pigment that supports them. The corrective MYO7A gene was introduced into RPE cells, thereby correcting Usher 1B syndrome. used as a gene therapy product designed to attenuate or reverse vision loss associated with The gene carried by the AAV vector encodes the MYO7A protein. MYO7a cDNA (a large gene over 100 mb in length). The expression of the A gene was controlled by the CMV promoter, the CMV / MYO7A chimeric promoter, or AAV vectors can be driven by alternative promoters. It may be administered by subretinal injection.

[0597] In another embodiment, the AAV vector of the invention comprises an ATP-binding cassette gene for correction. ABCA4 (also known as ABCR) is introduced into photoreceptor cells, thereby inhibiting Stargazing. AAV vectors can be used to attenuate or reverse the pathophysiology leading to Sclerosis. The gene carried by the ABCA4 gene is the ABCA4 cDNA that encodes the ABCA4 protein. The expression of the ABCA4 gene is driven by a CMV promoter, a photoreceptor-specific promoter, e.g. For example, the AAV vector may be driven by rhodopsin kinase or an alternative promoter. can be administered by direct subretinal injection after vitrectomy of the eye.

[0598] In another embodiment, the AAV vector of the present invention is used to treat wet age-related macular degeneration (AMD), diabetes, Abnormal blood vessel growth and / or retinal vein occlusion in the eyes of patients with pathological macular edema or retinal vein occlusion or to prevent recurrence of vascular leakage and / or dry age-related macular degeneration (AMD) and a gene therapy product designed to prevent abnormal blood vessel growth in the eyes of patients with This AAV vector can be used to express angiostatin and / or endostatin. One or more genes encoding one or more anti-angiogenic proteins, such as thrombin In one embodiment, the AAV vector is adapted for delivery to retinal pigment epithelial cells. Bicistronic human endostatin using an internal ribosome entry site (IRES) Expression of anti-angiogenic genes (one or more) that express the angiostatin and angiostatin genes Currently, CMV and RPE-specific promoters, such as vitelloid macular dystrophy 2 (VMD) 2) by a promoter (more recently known as the bestrophin promoter) The AAV vector may be driven by a promoter other than the promoter of interest, or by an alternative promoter. It may be administered by direct subretinal injection after excision.

[0599] In another embodiment, the AAV vector of the invention is administered by injecting (one or more) the AAV vector into the donor cornea prior to transplantation. Preventing corneal graft rejection as a result of neovascularization by delivery of (multiple) antiangiogenic genes In one embodiment, the AAV vector can be used as a gene therapy product designed to The nucleotide sequence of the ... The bicistronic human endostatin and angiostatin genes were used to The AAV vector expresses one or more anti-angiogenic genes. Transduced donor tissue may also be stored prior to transplantation. Expression of anti-angiogenic genes (one or more) is driven by a constitutive promoter such as the CMV promoter. However, it is possible that alternative promoters can be used. be.

[0600] In another embodiment, the AAV vector of the present invention is used to treat wet age-related macular degeneration (AMD), diabetes, Abnormal blood vessel growth and / or retinal vein occlusion in the eyes of patients with pathological macular edema or retinal vein occlusion or to prevent recurrence of vascular leakage and / or dry age-related macular degeneration (AMD) As a gene therapy designed to prevent abnormal blood vessel growth in the eyes of patients with This AAV vector encodes a soluble form of the fms-like tyrosine kinase. (Soluble Flt-1) The expression of the soluble Flt-1 gene is V, RPE-specific promoter, e.g., vitelloid macular dystrophy 2 (VMD2) promoter promoter (more recently known as the bestrophin promoter), and The AAV vector can be driven by an alternative promoter. It may be administered by subretinal injection.

[0601] In another embodiment, the AAV vector of the present invention...

Claims

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

2. A nucleic acid sequence comprising a nucleotide of interest and a TRAP binding site, (i) the TRAP binding site is located between the nucleotide of interest and the start codon ATG; a portion of the TRAP binding site, or the ATG start codon includes a portion of the TRAP binding site; and / or or (ii) the nucleic acid sequence also includes a Kozak sequence, and the Kozak sequence is linked to the TRAP binding site. Including part of the place Nucleic acid sequence.

3. The nucleotide of interest is operably linked to the TRAP binding site or a portion thereof.

3. The nucleic acid sequence of claim 1 or 2.

4. The TRAP binding site or a portion thereof is a sequence that allows translation of the nucleotide of interest to occur in a viral vector. Tryptophan RNA-binding attenuator protein (TARP) is expressed in the vector-producing cells, as it is suppressed in the vector-producing cells.

10. A nucleic acid sequence according to any preceding claim, capable of interacting with a protein.

5. The nucleotide of interest lacks the tryptophan RNA-binding attenuating protein.

10. A nucleic acid sequence according to any preceding claim, which is translated in a target cell.

6. The TRAP binding site or a portion thereof comprises multiple repeats of the sequence KAGN2-3.

10. A nucleic acid sequence according to any one of claims 1 to 9.

7. The preceding, wherein the TRAP binding site or a portion thereof comprises multiple repeats of the sequence KAGN2. A nucleic acid sequence according to any one of claims 1 to 4.

8. The TRAP binding site or part thereof comprises at least six repeats of the sequence KAGN2. , a nucleic acid sequence according to any of the preceding claims.

9. The TRAP binding site or a portion thereof contains at least eight repeats of the sequence KAGN2-3.

10. A nucleic acid sequence according to any preceding claim, comprising:

10. 10. The nucleic acid sequence of claim 9, wherein the number of KAGNNN repeats is one or less.

11. The TRAP binding site or a portion thereof contains at least 8 to 11 repeats of the sequence KAGN2.

10. A nucleic acid sequence according to any preceding claim, comprising:

12. The TRAP binding site or a portion thereof comprises 11 repeats of the sequence KAGN2-3, 10. The nucleic acid sequence of any preceding claim, wherein the number of GNNN repeats is 3 or less. Column.

13. The Kozak sequence and / or the start codon is or is a part of the TRAP binding site 10. A nucleic acid sequence according to any preceding claim, which overlaps with the 3' end of

14. The Kozak sequence and / or the start codon is or is a part of the TRAP binding site 14. The nucleic acid sequence of claim 13, which overlaps with the 3'-terminal KAGNN repeat of

15. The Kozak sequence is selected from the group consisting of the sequence RNNATG (SEQ ID NO: 125) and RVVATG (SEQ ID NO: 126). 28). A nucleic acid sequence according to any of the preceding claims, comprising:

16. The overlapping Kozak sequence and / or the initiation codon and the TRAP binding site or any one of SEQ ID NOs: 29 to 33. Nucleic acid sequence of.

17. The nucleic acid sequence is any one of SEQ ID NOs: 34-37, 69-92 or 108-112. 114).

18. 18. The nucleic acid of claim 17, wherein the nucleic acid sequence comprises one of SEQ ID NO: 34 or SEQ ID NO:

35. Acid sequence.

19. From the end of the transcription start site / promoter to the start of the TRAP binding site or part thereof 10. The nucleic acid sequence of any of the preceding claims, wherein the distance between said first and second nucleotides is from 1 to 33 nucleotides in length.

20. From the end of the transcription start site / promoter to the start of the TRAP binding site or part thereof 10. The nucleic acid sequence of any of the preceding claims, wherein the distance between said first and second nucleotides is 1 to 12 nucleotides in length.

21. The TRAP binding site or a part thereof is a type II restriction enzyme site, preferably a SapI site.

10. A nucleic acid sequence according to any preceding claim, which lacks restriction enzyme sites.

22. The nucleic acid sequence includes a 5' leader sequence upstream of the TRAP binding site or a portion thereof.

10. A nucleic acid sequence according to any one of the preceding claims.

23. 10. The method of claim 9, wherein the leader sequence comprises a sequence derived from the non-coding EF1α exon 1 region.

23. The nucleic acid sequence according to claim 22.

24. 2. The method of claim 1, wherein the leader sequence comprises the sequence defined in SEQ ID NO: 25 or SEQ ID NO:

26.

24. The nucleic acid sequence of claim 23.

25. 10. Any of the preceding claims, wherein the sequence comprises an internal ribosome entry site (IRES). A nucleic acid sequence as described above.

26. The sequence may comprise an internal ribosome entry site (IRES) and a TRAP binding site or both thereof.

26. The nucleic acid sequence of claim 25, comprising a spacer sequence between the two portions.

27. 27. The nucleic acid sequence of claim 26, wherein the spacer is 0 to 30 nucleotides in length.

28. 28. The nucleic acid sequence of claim 27, wherein the spacer is 15 nucleotides in length.

29. The spacer is located between the 3' end of the TRAP binding site or portion thereof and the target of interest. 26 to 27, wherein the nucleotide is 3 or 9 nucleotides downstream from the start codon.

9. A nucleic acid sequence according to any one of claims 8.

30. The spacer comprises a sequence defined in any one of SEQ ID NOs: 38 to 44, preferably any one of claims 26 to 29, wherein the spacer comprises the sequence defined in SEQ ID NO: 39 1. The nucleic acid sequence of claim 1.

31. 10. The method of claim 1, wherein the nucleotide of interest produces a therapeutic effect. The nucleic acid sequence described above.

32. Any of the preceding claims, wherein the nucleic acid sequence further comprises an RRE sequence or a functional substitute thereof. A nucleic acid sequence according to any one of claims 1 to 4.

33. 10. The method of claim 9, wherein the nucleic acid sequence is a vector transgene expression cassette. The nucleic acid sequence described.

34. The 3'-terminal KAGNN repeat of the TRAP binding site or part thereof comprises at least 10. The method of claim 9, wherein the first nucleotide of the initiation codon ATG overlaps with the first nucleotide of the initiation codon ATG. The nucleic acid sequence described above.

35. The 3'-end KAGNN repeat of or a portion of the TRAP binding site is 35. The nucleic acid sequence of claim 34, which overlaps the first two nucleotides of the donor ATG.

36. The 3'-terminal KAGNN repeat of or part of the TRAP binding site is 35. The method of claim 34, wherein the first nucleotide of the initiation codon ATG in the nucleotide sequence overlaps with the first nucleotide of the initiation codon ATG in the nucleotide sequence. Nucleic acid sequence.

37. The nucleic acid sequence comprises the sequence defined in SEQ ID NO: 114 or SEQ ID NO:

116.

37. A nucleic acid sequence according to any one of paragraphs 34 to 36.

38. A viral vector comprising a nucleic acid sequence according to any one of claims 1 to 37 or 67 to 92. Tar.

39. The viral vector contains more than one nucleotide of interest and at least one The nucleotide of interest is a TRAP binding site as defined in any one of claims 1 to 12. The viral vector of claim 38, wherein the viral vector is operably linked to a site or portion thereof.

40. The viral vector may be a retrovirus, an adenovirus, an adeno-associated virus, or a simple virus.

3. The vector according to claim 3, which is derived from a pure herpesvirus, a vaccinia virus, or a baculovirus. 8 or 39. A viral vector according to claim 39.

41. The viral vector is derived from a lentivirus, preferably the viral vector is HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or Visnare 41. The viral vector of claim 40, which is derived from an antivirus.

42. A viral vector comprising a set of nucleic acid sequences encoding components required for the production of a viral vector. A viral vector production system, wherein the RNA genome of the viral vector is 7 or 67 to 92. A viral vector production system comprising the nucleic acid sequence of any one of

43. The viral vector is a retrovirus, an adenovirus, or an adeno-associated virus. Preferably, the viral vector is a retroviral vector, and the viral vector is derived from The virus vector production system contains Gag and Pol proteins, the tryptophan RNA binding site, Encoding a fusion attenuation protein and an Env protein, or functional substitutes thereof 43. The viral vector production system of claim 42, comprising a nucleic acid sequence.

44. The viral vector production system comprises a nucleic acid sequence encoding rev or a functional substitute thereof.

44. The viral vector production system of claim 43, further comprising:

45. Preferably, said viral vector is derived from a lentivirus. HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or Visna The production of a viral vector according to any one of claims 42 to 44, which is derived from a lentivirus. system.

46. Claims 42 to 4, comprising a nucleic acid sequence according to any one of claims 1 to 37 or 67 to 92 6. A DNA construct for use in the viral vector production system according to any one of 5.

47. The method of claim 1, further comprising: A DNA construct for use in a viral vector production system according to any one of 42 to 45. construction.

48. 48. The DNA construct of claim 46 or claim 47, and Gag and Pol proteins. and a DNA construct encoding the Env protein or a functional substitute thereof. The viral vector production system according to any one of claims 42 to 45, comprising a NA construct. A set of DNA constructs for use in the preparation of a medicament for the treatment ... a DNA construct further comprising a DNA construct encoding a rev sequence or a functional substitute thereof. A set of things.

49. The nucleic acid sequence according to any one of claims 1 to 37 or 67 to 92, any one of claims 42 to 45 A viral vector production system according to any one of claims 446 to 48 or D Viral vector-producing cells containing the NA construct.

50. The cells are transiently transfected with a vector encoding a tryptophan-RNA binding attenuator protein.

50. The viral vector producing cell of claim 49, which is transfected transfectant.

51. 4. The cell stably expresses a tryptophan-RNA binding attenuator protein.

10. A viral vector producing cell according to 9.

52. The nucleic acid sequence according to any one of claims 1 to 37 or 67 to 92, any one of claims 42 to 45 A viral vector production system according to any one of claims 46 to 48 or a DNA according to any one of claims 46 to 48. Introducing the A construct into a viral vector-producing cell and producing the viral vector. and culturing the producer cells under conditions suitable for producing the viral vector. How to do this.

53. A method for producing a viral vector by using the viral vector-producing cell according to any one of claims 49 to 51 or claim The viral vector according to any one of claims 42 to 45, which is produced by the method according to claim 52. Viral vectors produced by production systems.

54. The nucleic acid sequence according to claim 53, comprising the nucleic acid sequence according to any one of claims 1 to 37 or 67 to 92. The viral vectors described above.

55. 53 or 54, derived from a retrovirus, an adenovirus or an adeno-associated virus. Or a viral vector described in claim 54.

56. 56. The viral vector of claim 55, which is derived from a lentivirus.

57. HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or Visna 57. The viral vector of claim 56, which is derived from a lentivirus.

58. By the viral vector according to any one of claims 38 to 41 or 53 to 57 Transduced cells.

59. 58. A composition according to any one of claims 38 to 41 or 53 to 57 for use in medicine. A viral vector or a cell according to claim 58.

60. Delivering a nucleotide of interest to a target site where the nucleotide of interest is required.

58. The method of claim 38, 41 or 53, 57 for the preparation of a medicament for 60. Use of a viral vector or a cell according to claim 58.

61. A viral vector or a virus vector according to any one of claims 38 to 41 or 53 to 57. A method of treatment comprising administering the cells of paragraph 58 to a subject in need thereof.

62. Claims 38 to 41 in combination with a pharmaceutically acceptable carrier, diluent or excipient. or the viral vector according to any one of claims 53 to 57 or the cell according to claim 58 10. A pharmaceutical composition comprising:

63. The translation of the nucleotide of interest when operably linked to a nucleic acid binding site is inhibited by the viral vector. nucleic acid binding sites that can interact with the receptor so as to be inhibited in receptor-producing cells; and / or a method for identifying a nucleic acid binding protein operably linked to a reporter gene. A reporter gene in a cell containing both the nucleic acid binding site and the nucleic acid binding protein is The method comprises analyzing expression of a target gene.

64. 64. The method of claim 63, wherein the reporter gene encodes a fluorescent protein.

65. Repressing translation of a nucleotide of interest (NOI) in viral vector producing cells 10. A method for detecting a nucleic acid sequence as defined in any one of claims 1 to 37 or 67 to 92. , and a nucleic acid sequence encoding tryptophan-RNA binding decay protein (TRAP) into the viral vector-producing cells, A method of binding to a binding site or part thereof, thereby inhibiting translation of said NOI.

66. A method for increasing viral vector titer in eukaryotic vector-producing cells, a virus according to any one of claims 42 to 45 in the eukaryotic vector-producing cell; A gene vector production system and tryptophan-RNA binding decay protein (TRAP) coding the TRAP comprises introducing a nucleic acid sequence encoding the TRAP binding site or and inhibits translation of said NOI, thereby inhibiting translation of a gene that does not have a TRAP binding site. The method of claim 1, wherein the viral vector titer is increased relative to a viral vector.

67. The nucleotide of interest and tryptophan RNA-binding decay protein (TRAP) a nucleic acid sequence comprising a binding site for a nucleotide sequence encoding ... The multiple cloning site is 3' of the TRAP binding site. ２～３ Repetition and overlap or the 3' KAGN of the TRAP binding site ２～３ Downstream and above for the repeat A nucleic acid sequence located upstream of a Kozak sequence.

68. The nucleic acid sequence of claim 67, wherein the nucleic acid sequence comprises any one of SEQ ID NOs: 45 to 58. 。

69. The nucleic acid sequence of claim 68, wherein the nucleic acid sequence comprises any one of SEQ ID NOs: 52 to 58. 。

70. the nucleic acid sequence comprises any one of SEQ ID NO:52, SEQ ID NO:55, or SEQ ID NO:58 70. The nucleic acid sequence of claim 69.

71. The nucleic acid sequence of claims 1 to 37 or 6 further comprises a promoter-5'UTR region.

71. A nucleic acid sequence according to any one of claims 7 to 70.

72. the TRAP binding site or a part thereof and a Kozak sequence, or the TRAP binding site a multiple cloning site and a Kozak sequence in the promoter-5'UTR region 72. The nucleic acid sequence of claim 71, located within the 5'UTR.

73. The promoter-5'UTR region further comprises an intron, preferably 72. A method according to claim 71, wherein a nucleotide sequence is upstream of the TRAP binding site or a portion thereof. The nucleic acid sequence described in

74. The promoter-5'UTR region includes a heterologous intron within the 5'UTR.

74. The nucleic acid sequence of any one of claims 71 to 73, which is a promoter.

75. A nucleic acid sequence encoding the RNA genome of a viral vector, said viral vector - the RNA genome of any one of claims 1 to 37 or 67 to 74 A nucleic acid sequence, including a sequence.

76. 38. The method of claim 1, wherein the nucleic acid sequence is contained within the RNA genome of a viral vector. 67 to 74. A nucleic acid sequence according to any one of claims 67 to 74.

77. the nucleic acid sequence is a nucleotide sequence encoding the RNA genome of a viral vector A nucleic acid according to any one of claims 1 to 37 or 67 to 74, which is functionally linked sequence. **Claim 78** The major splice donor site in the RNA genome of the viral vector is inactivated A nucleic acid sequence according to any one of claims 75 to 77 or a viral vector production system according to any one of claims 43 to 45. **Claim 79** In the RNA genome of the viral vector, the major splice donor site and the Potential splice donor sites 3' to the major splice donor site are inactivated And preferably the potential splice donor site is the first potential splice donor site 3' to the major splice donor site. A nucleic acid sequence or viral vector production system according to claim 78. **Claim 80** The potential splice donor site is within 6 nucleotides of the major splice donor site. A nucleic acid sequence or viral vector production system according to claim 75. **Claim 81** The major splice donor site and potential splice donor sites are mutated or deleted. A nucleic acid sequence or viral vector production system according to any one of claims 78 to 80. **Claim 82** The nucleotide sequence encoding the RNA genome of the viral vector before inactivation of the splice site Contains a sequence shown in any of SEQ ID NOs: 94, 96, 97, 102, 103 and / or 106. A nucleic acid sequence or viral vector production system according to any one of claims 78 to 81. **Claim 83** The nucleotide sequence encoding the RNA genome of the viral vector contains a sequence having a mutation or deletion with respect to the sequence shown in any of SEQ ID NOs: 94, 96, 97, 102, 103 and / or 106. A nucleic acid sequence or viral vector production system according to any one of claims 78 to 82. **Claim 84** The nucleotide sequence encoding the RNA genome of the viral vector contains an inactivated major splice donor site having a cleavage site between nucleotides corresponding to nucleotides 13 and 14 of SEQ ID NO: 94 if not inactivated. A nucleic acid sequence or viral vector production system according to any one of claims 78 to 83. [[ID=३३]] **Claim 85** The nucleotide sequence of the major splice donor site before inactivation is SEQ ID NO: 97 85. The nucleic acid sequence or virus according to any one of claims 78 to 84, comprising the sequence shown in Vector production system.

86. The nucleotide sequence of the cryptic splice donor site before inactivation is SEQ ID NO:1 86. The nucleic acid sequence according to any one of claims 79 to 85, comprising the sequence shown in Virus vector production system.

87. The nucleotide sequence encoding the RNA genome of the viral vector is If not activated, the nucleotides corresponding to nucleotides 17 and 18 of SEQ ID NO:94 79 to 79, comprising an inactivated cryptic splice donor site with a cleavage site therebetween.

87. A nucleic acid sequence or viral vector production system according to any one of claims 86.

88. The nucleotide sequence encoding the RNA genome of the viral vector is Any of numbers 95, 98, 99, 100, 101, 104, 105 and / or 107 88. A nucleic acid sequence or virus according to any one of claims 78 to 87, comprising the sequence shown in rus vector production system.

89. The nucleotide sequence encoding the RNA genome of the viral vector is The nucleic acid sequence according to any one of claims 78 to 88, which does not include the sequence shown in number 102. Array or viral vector production system.

90. The major splice donor site and cryptic splice donor site of the RNA genome of the viral vector 78. The method of claim 78, wherein splicing activity from an alternative splice donor site is inhibited or eliminated.

90. A nucleic acid sequence or viral vector production system according to any one of claims 1 to 89.

91. The major splice donor site and cryptic splice donor site of the RNA genome of the viral vector Splicing activity from the alternative splice donor site was also observed in transfected cells. is suppressed or ablated in the transduced cells according to any one of claims 78 to 90 A nucleic acid sequence or viral vector production system according to claim 1.

92. 92. Any one of claims 67 to 91, wherein the viral vector is derived from a lentivirus. A nucleic acid sequence or viral vector production system according to claim 1.

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