Retroviral vectors

Abstract

The present invention relates to retroviral vectors, particularly lentiviral vectors, which contain modified retroviral RNA sequences with codon substitutions and a reduced number of retroviral open reading frames, and which are pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxoviruses, methods for making same, and uses thereof.

Description

[Technical Field]

[0001] The present invention relates to retroviral vectors, particularly lentiviral vectors, which contain modified retroviral RNA sequences with codon substitutions and a reduced number of retroviral open reading frames, and which are pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxoviruses, methods for making same, and uses thereof. [Background technology]

[0002] Retroviruses are a family of RNA viruses (Retroviridae) that encode the enzyme reverse transcriptase. Lentiviruses are a genus of the Retroviridae family and are characterized by a long latency period. Retroviruses, particularly lentiviruses, have the unique ability among retroviruses to deliver significant amounts of viral RNA into the DNA of host cells and to infect non-dividing cells, making them one of the most efficient gene delivery vectors.

[0003] Pseudotyping is the process of generating a virus or viral vector by combining it with a foreign viral envelope protein. Therefore, the foreign viral envelope protein can be used to alter the host tropism or increase / decrease the stability of the viral particle. For example, pseudotyping allows for defining the characteristics of the envelope protein. A protein frequently used to pseudotype retroviral and lentiviral vectors is the glycoprotein G of vesicular stomatitis virus (VSV), abbreviated as VSV-G.

[0004] Lentiviral vectors, especially those derived from HIV-1, are widely studied and frequently used vectors.The evolution of lentiviral vector backbone and the ability of viruses to deliver recombinant DNA molecules (transgenes) to target cells have led to their use in many applications.Two possible applications of viral vectors include functional gene repair in gene therapy and in vitro recombinant protein production.

[0005] When designing suitable retroviral / lentiviral vectors for use as gene delivery vectors, one important driver is to make the vector as safe as possible for patients.The second important driver is the need to produce a sufficient amount of vector, not only to treat individual patients, but also to allow wider clinical access to therapy for all patients who may benefit from therapy.These two drivers can be found to be contradictory, because the modification that improves the safety of vectors is often associated with a decrease in yield during vector production.

[0006] One example of a clinical situation that would benefit from gene transfer into airway epithelia is the treatment of cystic fibrosis (CF). CF is a fatal genetic disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which acts as a chloride channel in airway epithelial cells. CF is characterized by recurrent chest infections, increased airway secretions, and ultimately respiratory failure. In the UK, the current median age at death is approximately 25 years. For most genotypes, no treatments target the underlying defect, and current treatments for symptomatic relief require self-administered therapy for several hours daily. Unlike small molecule drugs, gene therapy is independent of CFTR mutation class and is therefore applicable to all affected CF individuals. However, to date, no viral vectors have been approved for clinical use in the treatment of CF; the same is true for other diseases, particularly many other respiratory tract diseases.

[0007] In addition to patient safety and yield issues, there are other difficulties traditionally associated with gene transfer to airway epithelia.

[0008] Gene transfer efficiency into respiratory epithelia is generally low, at least in part because the respective receptors for many viral vectors appear to be primarily localized on the basolateral surface of the airway epithelium. Therefore, prior to our studies, the use of lentiviral pseudotypes required disruption of epithelial integrity to transduce the airways, for example, by the use of detergents such as lysophosphatidylcholine or ethylene glycol bis(2-aminoethyl ether)-N,N,N'N'-tetraacetic acid, and was associated with an increased risk of sepsis. In addition, conventional gene transfer vectors struggle to penetrate the respiratory airway mucus layer, which also reduces gene transfer efficiency. The ability to repeatedly administer conventional viral vectors, which is essential for lifelong treatment of self-renewing epithelia, is limited due to the patient's adaptive immune response, which prevents successful repeated administration.

[0009] Administration of vectors for clinical use is another relevant factor: therefore, viral stability through the use of clinically relevant devices (e.g., bronchoscopes and nebulizers) must be maintained for therapeutic efficacy. Summary of the Invention [Problem to be solved by the invention]

[0010] Thus, there is a need for gene therapy vectors that can avoid one or more of the problems described above. In particular, it is an object of the present invention to provide methods and means for producing pseudotyped retroviral or lentiviral (e.g., SIV) vectors, and means for carrying out said methods, wherein the resulting vectors are safe, adapted for improved gene transfer efficiency across respiratory epithelia, and produced on a clinically relevant scale. [Means for solving the problem]

[0011] The present inventors have previously developed lentiviral vectors pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxoviruses, containing a promoter and a transgene. Typically, the vector backbone is derived from simian immunodeficiency viruses (SIVs), such as SIV1 or African green monkey SIV (SIV-AGM). Preferably, the backbone of the viral vectors of the present invention is derived from SIV-AGM. The HN and F proteins, respectively, attach to sialic acid and function to mediate cell fusion for vector entry into target cells. The present inventors have discovered that this specifically F / HN-pseudotyped lentiviral vector can efficiently transduce airway epithelia, resulting in sustained transgene expression for a period exceeding the intended lifespan of the airway epithelial cells. Importantly, the present inventors have also found that re-administration does not result in a loss of efficacy. These features make the vectors of the present invention attractive candidates for treating disease through their use in expressing therapeutic proteins that are (i) intracellular in the respiratory tract; (ii) secreted into the lumen of the respiratory tract; and (iii) secreted into the circulatory system.

[0012] However, there have been potential safety concerns with this lentiviral vector. In particular, lentiviral vectors contain a significant number of retroviral (i.e., non-transgene) open reading frames (ORFs). There is a theoretical risk that the retroviral ORFs may be expressed after administration to patients. The expression of retroviral ORFs represents a safety risk to patients, especially if the patient has an immune response to the expressed retroviral sequences.

[0013] Furthermore, the significant degree of sequence homology between the retroviral vector and the GagPol plasmid used in production creates the additional theoretical risk that replication-competent lentivirus (RCL) may arise either during manufacturing or in clinical use after administration to patients. This represents an additional safety risk for patients. The risk of generating replication-competent viral particles is similarly an issue for other retroviral / lentiviral vectors.

[0014] While it is desirable to mitigate these risks, doing so is not straightforward, or at least not without incurring other unacceptable drawbacks. On the other hand, modifications to reduce the number of ORFs, particularly those 5′ to a promoter-transgene, risk affecting the expression of downstream transgenes. Furthermore, other modifications to retroviral genomes, such as codon substitutions to introduce stop codons to reduce the length of retroviral ORFs, can also have deleterious effects on, for example, vector yield and / or transgene expression. Additionally, modifications aimed at reducing the risk of RCL, such as codon optimization of the gag-pol gene to be produced, are known in the art to typically negatively impact vector titer or yield. Given that large titers of vector are required to treat even a single patient, such reductions in yield have the potential to render their production commercially unviable.

[0015] As described herein, the inventors have designed and generated retroviral vectors, particularly SIV vectors, containing retroviral RNA sequences modified to reduce the number of retroviral ORFs and introduce specific codon substitution modifications. The modified retroviral vectors of the present invention, containing these newly described retroviral RNA sequences, mitigate one or more of the risks described above and provide clinically advantageous products. Furthermore, the inventors have surprisingly demonstrated that benefits can be obtained without expected disadvantages, such as reduced transgene expression and / or reduced vector yield. While such modifications have previously been considered in the context of proviral DNA, this application is the first to elucidate these modifications within the retroviral / lentiviral RNA sequence itself, rather than within a manufacturing platform. Furthermore, this application is the first to demonstrate the benefits conferred by specific modifications to a retroviral / lentiviral RNA sequence, and to show that this extends not only to beneficial effects on vector yield, but also to transgene expression and integration of the retroviral / lentiviral RNA sequence into host / target cells.

[0016] Specifically, the inventors identified potential SIV ORFs within the SIV RNA sequence. The SIV RNA sequence was modified to remove one or more SIV ORFs. Specifically, the inventors removed one or more SIV ORFs located 5' to the transgene promoter, one or more SIV ORFs encoding polypeptides greater than or equal to 100 amino acids in length, one or more ORFs contained (at least in part) in a partial RRE sequence, and / or one or more ORFs contained (at least in part) in a partial Gag sequence. Removal of SIV ORFs was achieved by removing the start codon (ATG) of the selected SIV ORF. To determine which SIV ORFs (and combinations thereof) could be removed without affecting downstream transgene expression, the inventors generated several different SIV vectors. Each SIV vector was evaluated to quantify vector yield and transgene expression of the modified SIV vector and the corresponding unmodified vector.

[0017] We demonstrated that the aforementioned modifications (both codon substitutions and modifications to reduce the number of SIV ORFs) do not negatively affect transgene expression by SIV vectors pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxoviruses and can even result in increased transgene expression by the vectors, which is surprising given that it is generally accepted that such modifications, while addressing potential safety concerns, can have deleterious effects on transgene expression.

[0018] Additionally, the aforementioned mutations (both codon substitutions and modifications to reduce the number of SIV ORFs) have no negative effect on, and may even result in increased integration of, SIV vectors pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxoviruses into host / target cells. Again, this is surprising given that it is generally accepted that such modifications, while addressing potential safety issues, may have deleterious effects on vector integration.

[0019] Furthermore, the aforementioned mutations (both codon substitutions and modifications to reduce the number of SIV ORFs) have no negative impact on the yield of SIV vectors pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxoviruses and can even result in increased vector titers. Again, this is surprising given that it is generally accepted that such modifications, while addressing potential safety issues, can have deleterious effects on vector yield.

[0020] Thus, the present invention provides a retroviral vector comprising a modified retroviral RNA sequence that (i) has codon substitutions and (ii) contains a reduced number of retroviral open reading frames (ORFs) compared to the unmodified retroviral RNA sequence from which it is derived, wherein (a) the retroviral RNA sequence comprises a promoter and a transgene; and (b) the retroviral vector is pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus.

[0021] Also disclosed are methods for the generation of retroviruses, particularly lentiviral vectors such as SIV, comprising a retroviral RNA sequence that has been codon substituted and contains a reduced number of retroviral OFRs compared to the unmodified plasmid genome vector from which the modified retroviral genomic RNA sequence is derived, wherein (a) the retroviral RNA sequence comprises a promoter and a transgene, and (b) the retroviral vector is pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus, thereby reducing the risk of an immune response when administered to a patient without negatively affecting transgene expression.

[0022] The modified retroviral genomic RNA sequence may lack (a) one or more retroviral ORFs 5' of the promoter; (b) one or more retroviral ORFs encoding a polypeptide ≥ 100 amino acids in length; (c) one or more retroviral ORFs comprised (at least in part) in the partial RRE sequence; and / or (d) one or more retroviral ORFs comprised (at least in part) in the partial Gag sequence.

[0023] The respiratory paramyxovirus may be a Sendai virus.

[0024] The promoter may be selected from the group consisting of a hybrid human CMV enhancer / EF1a (hCEF) promoter, a cytomegalovirus (CMV) promoter, and an elongation factor 1a (EF1a) promoter. Preferably, the vector comprises a hybrid human CMV enhancer / EF1a (hCEF) promoter.

[0025] The transgene may be selected from (a) CFTR, ABCA3, DNAH5, DNAH11, DNAI1, and DNAI2; or (b) a secreted therapeutic protein, optionally alpha-1 antitrypsin (A1AT), factor VIII, surfactant protein B (SFTPB), factor VII, factor IX, factor X, factor XI, von Willebrand factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), and a monoclonal antibody against an infectious agent. Preferably, the transgene may encode (a) CFTR; (b) A1AT; or (c) FVIII.

[0026] The promoter may be an hCEF promoter and the transgene may encode CFTR. The promoter may be an hCEF promoter and the transgene may encode A1AT. The promoter may be an hCEF or CMV promoter and the transgene may encode FVIII.

[0027] The retroviral vector may be a lentiviral vector, optionally selected from the group consisting of an SIV vector, a human immunodeficiency virus (HIV) vector, a feline immunodeficiency virus (FIV) vector, an equine infectious anemia virus (EIAV) vector, and a Visna / Maedi virus vector. Preferably, the retroviral vector is an SIV vector.

[0028] The modified retroviral RNA sequence may (i) be less than 9,000 bases in length, and / or (ii) comprise or consist of a nucleic acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% identity to SEQ ID NO: 1. Preferably, the modified retroviral RNA sequence may (i) be less than 9,000 bases in length, and (ii) comprise or consist of a nucleic acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% identity to SEQ ID NO: 1. More preferably, the modified retroviral RNA sequence may comprise or consist of the nucleic acid sequence of SEQ ID NO:1, and even more preferably, the modified retroviral RNA sequence may consist of the nucleic acid sequence of SEQ ID NO:1.

[0029] The retroviral vector comprises: (a) a p17 protein comprising or consisting of an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity to SEQ ID NO:2; (b) a p17 protein having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity to SEQ ID NO:3; (c) a p24 protein comprising or consisting of an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity to SEQ ID NO:4; (d) a p8 protein comprising or consisting of an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to SEQ ID NO:5; (e) a protease comprising or consisting of an amino acid sequence having 99.9%, or at most 100%, sequence identity to SEQ ID NO:6; (f) a p51 protein comprising or consisting of an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or at most 100% sequence identity to SEQ ID NO:7; and / or (g) a p31 protein comprising or consisting of an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity to SEQ ID NO: 8. Optionally, the vector may comprise each of (a) to (g).

[0030] The retroviral vector may further comprise one or more of: (a) a Gag protein comprising, or consisting of, an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity to SEQ ID NO:9; and / or (b) a Pol protein comprising, or consisting of, an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity to SEQ ID NO:10.

[0031] The present invention also provides an SIV vector pseudotyped with Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) proteins, wherein (a) the vector comprises a modified retroviral RNA sequence comprising or consisting of the nucleic acid sequence of SEQ ID NO: 1, preferably the modified retroviral RNA sequence consisting of the nucleic acid sequence of SEQ ID NO: 1; and (b) the F protein comprises a first subunit comprising or consisting of the amino acid sequence of SEQ ID NO: 14, and a second subunit comprising or consisting of the amino acid sequence of SEQ ID NO: 15. The vector may further comprise one or more of: (a) a p17 protein comprising or consisting of the amino acid sequence of SEQ ID NO:2; (b) a p24 protein comprising or consisting of the amino acid sequence of SEQ ID NO:3; (c) a p8 protein comprising or consisting of the amino acid sequence of SEQ ID NO:4; (d) a protease comprising or consisting of the amino acid sequence of SEQ ID NO:5; (e) a p51 protein comprising or consisting of the amino acid sequence of SEQ ID NO:6; (f) a p15 protein comprising or consisting of the amino acid sequence of SEQ ID NO:7; (g) a p31 protein comprising or consisting of the amino acid sequence of SEQ ID NO:8; (h) a Gag protein comprising or consisting of the amino acid sequence of SEQ ID NO:9; and / or (i) a Pol protein comprising or consisting of the amino acid sequence of SEQ ID NO:10; optionally, the vector comprises each of (a) to (g).

[0032] Also disclosed are methods for producing retroviruses, particularly lentivirus vectors such as SIV, comprising a retroviral RNA sequence that has undergone codon substitutions and contains a reduced number of retroviral OFRs compared to the unmodified plasmid genome vector from which the modified retroviral genomic RNA sequence is derived, wherein (a) the retroviral RNA sequence comprises a promoter and a transgene, and (b) the retroviral vector is pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus, thereby reducing the risk of RCL without negatively affecting or even increasing vector titer, vector integration, and / or transgene expression. Thus, the methods of the present invention provide safer vectors produced in commercially desirable yields.

[0033] Thus, the present invention also provides a method for producing a retroviral vector that contains codon substitutions and a reduced number of ORFs compared to the unmodified retroviral RNA sequence from which the modified retroviral RNA sequence is derived, wherein the retroviral RNA sequence comprises a promoter and a transgene and is pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus. The method may comprise or consist of the following steps: (a) growing cells in suspension; (b) transfecting the cells with one or more plasmids; (c) adding a nuclease; (d) harvesting the lentivirus; (e) adding trypsin (or an enzyme with the same cleavage specificity); and (d) purifying.

[0034] Steps (a) to (f) of the method may be performed sequentially. The cells may be HEK293 cells (such as HEK293F or HEK293T cells) or 293T / 17 cells. The addition of nuclease may be a pre-harvest step. The addition of trypsin (or an enzyme with the same cleavage specificity) may be a post-harvest step. The purification step may include one or more chromatography steps.

[0035] The present invention further provides a retroviral vector which has codon substitutions and a reduced number of ORFs compared to the unmodified retroviral RNA sequence from which the modified retroviral RNA sequence is derived, wherein the retroviral RNA sequence comprises a promoter and a transgene and is pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus, and which can be obtained by the method of the present invention.

[0036] The present invention also provides a composition comprising a retroviral vector and a pharmaceutically acceptable excipient or diluent, wherein the retroviral vector comprises a modified retroviral RNA sequence that has codon substitutions and a reduced number of open reading frames compared to the unmodified retroviral RNA sequence from which the modified retroviral RNA sequence is derived, the retroviral RNA sequence comprising a promoter and a transgene, and the retroviral vector is pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus. The composition may be formulated for pulmonary administration; optionally, administration is by intratracheal or intranasal instillation, aerosol delivery, intravenous injection, or direct injection into the lungs.

[0037] The present invention also provides a retroviral vector for use in a method of treatment, wherein the retroviral vector comprises a modified retroviral RNA sequence that has codon substitutions and contains a reduced number of ORFs compared to the unmodified retroviral RNA sequence from which the modified retroviral RNA sequence is derived, the retroviral RNA sequence comprising a promoter and a transgene, and the retroviral vector is pseudotyped with the hemagglutinin-neuraminidase (HN) and fusion (F) protein from a respiratory paramyxovirus. The present invention also provides a method of treating a disease, comprising the step of administering a retroviral vector to a subject in need thereof, wherein the retroviral vector comprises a modified retroviral RNA sequence that has codon substitutions and contains a reduced number of ORFs compared to the unmodified retroviral RNA sequence from which the modified retroviral RNA sequence is derived, the retroviral RNA sequence comprising a promoter and a transgene, and the retroviral vector is pseudotyped with the hemagglutinin-neuraminidase (HN) and fusion (F) protein from a respiratory paramyxovirus. The disease to be treated may be a pulmonary disease, preferably cystic fibrosis. [Brief explanation of the drawings]

[0038] [Figure 1A] ~ [Figure 1G] 1A-F show schematic diagrams of exemplary plasmids used for the generation of vectors of the invention, with G representing the unmodified vector genome plasmid. [Figure 2] FIG. 2 shows a schematic diagram of an exemplary pDNA1 plasmid used for the generation of the A1AT vector of the present invention. [Figure 3A] ~ [Figure 3D] 3A-D show a schematic diagram of an exemplary pDNA1 plasmid used for the generation of a FVIII vector of the present invention. [Figure 4]There are 14 ATG start codons present in the Gag-RRE region of the pGM326 genomic plasmid that can result in ORFs longer than 10 amino acids. Arrows represent the ORFs that can arise from each of the labeled start codons. Circular ATGs are those that have a strong Kozak sequence and are in-frame with Gag or Env. [Figure 5] SIV-CFTR titers (TU / mL) of LVs produced using the Ambr® 15 bioreactor system as assessed by A549 FACS assay. VRC = Vector Reference Control [Figure 6] SIV-CFTR titers (TU / mL) of LV produced using the Ambr® 15 bioreactor system as assessed by a 3-day integration assay in HEK293T. Open bars indicate values ​​below the lower limit of quantitation. VRC = vector reference control. DNA extracted from cells harvested at 3 days was size-selection purified to remove unintegrated DNA and subjected to qPCR analysis. [Figure 7] A549 cells expressing CFTR protein as a percentage of the viable single-cell population analyzed by FACS. VRC = vector reference control; samples were diluted 1:20. [Figure 8] Western blot (using anti-PIV1 antibody ab20791 at a dilution of 1:5000) shows cleavage of Fct4 by the trypsin-like enzyme TrypLE. DETAILED DESCRIPTION OF THE INVENTION

[0039] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20th ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide those skilled in the art with a general dictionary of many of the terms used in this disclosure. The meaning and scope of the terms should be clear; however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. It should be understood that the present invention is not limited to the specific methodology, protocols, and reagents described herein, as these may vary.

[0040] The present disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0041] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Specific embodiments of, and examples for, the present disclosure are described herein for illustrative purposes; however, as those skilled in the relevant art will recognize, various equivalent modifications are possible within the scope of the present disclosure. For example, while method steps or functions are presented in a given order, alternative embodiments may perform the functions in a different order, or the functions may be performed substantially simultaneously. The teachings of the present disclosure provided herein may be applied to other procedures or methods, as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified, where necessary, to employ the compositions, functions, and concepts of the above references and applications to provide still further embodiments of the present disclosure. Also, due to considerations of biological functional equivalence, some changes can be made in protein structure without affecting biological or chemical action in type or amount. These and other modifications can be made to the present disclosure in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0042] Unless otherwise indicated, any nucleic acid sequence is written left to right in 5' to 3' orientation; an amino acid sequence is written left to right in amino to carboxy orientation, respectively.

[0043] The headings provided herein are not limitations of the various aspects or embodiments of the disclosure.

[0044] As used herein, the term "capable of" when used with a verb includes or means the action of the corresponding verb. For example, "capable of interacting" also means interacting, "capable of cleaving" also means cleaving, "capable of binding" also means binding, and "capable of specifically targeting" also means specifically targeting.

[0045] Other definitions of terms may appear throughout this specification. Before describing exemplary embodiments in more detail, it should be understood that the present disclosure is not limited to the particular embodiments described, as such may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure is defined solely by the appended claims.

[0046] Numerical ranges are inclusive of the numbers defining the range. When a range of values ​​is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range is also specifically disclosed, unless the context clearly dictates otherwise. Each smaller range between any stated or intervening value in a stated range and any other stated or intervening value within that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either or both limits are included in the smaller range, or neither limit is included in the smaller range, is also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included within the disclosure.

[0047] As used herein, the articles "a" and "an" may refer to one or to more than one (e.g., at least one) of the object of the article. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of "or" means "and / or" unless expressly stated otherwise. Furthermore, the use of the term "including" and other forms such as "includes" and "included" is not limiting.

[0048] "About" can generally refer to an acceptable degree of error for the quantity being measured, given the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically within 5% of a given value or range of values. Preferably, the term "about" is understood herein as plus or minus (±) 5% of the numerical value of the number with which it is used, preferably ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.1%.

[0049] The term "consisting of" refers to the compositions, methods, and their respective components described herein, to the exclusion of any element not recited in that description of the invention.

[0050] As used herein, the term "consisting essentially of" refers to elements required for a given invention. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristics of the invention (i.e., inert or non-immunogenic components).

[0051] Any embodiment described herein as "comprising" one or more features may also be considered a disclosure of the corresponding embodiment "consisting of" and / or "consisting essentially of" such features.

[0052] Concentrations, amounts, volumes, percentages, and other numerical values ​​may be presented herein in a range format. It should also be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values ​​explicitly recited as range limits, but also all individual numerical values ​​or subranges subsumed within the ranges as if each numerical value and subrange were explicitly recited.

[0053] As used herein, the terms "vector," "retroviral vector," and "retroviral F / HN vector" are used interchangeably to refer to a retroviral vector comprising a retroviral RNA sequence and pseudotyped with the hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus, unless otherwise stated. The terms "lentiviral vector" and "lentiviral F / HN vector" are used interchangeably to refer to a lentiviral vector pseudotyped with the hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus, unless otherwise stated. All disclosures herein regarding the retroviral vectors of the present invention apply equally and unconditionally to the lentiviral vectors of the present invention and to SIV vectors pseudotyped with the hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus (also referred to herein as SIV F / HN or SIV-FHN).

[0054] As defined herein, the term "retroviral RNA sequence" refers to a nucleic acid molecule contained within a retroviral vector. The retroviral RNA sequence includes a long terminal repeat (LTR) element, which is a nucleic acid sequence necessary for the incorporation of the retroviral RNA sequence into a retroviral particle and a transgene expression cassette. The transgene expression cassette is composed of a suitable enhancer / promoter element, a transgene cDNA, and post-transcriptional regulatory elements. The retroviral RNA sequence begins essentially with a 5' LTR R sequence and ends essentially with a 3' LTR R sequence. The retroviral RNA sequence in the 5' region typically comprises or consists (in 5' to 3' order) of a retroviral LTR R sequence followed by a retroviral LTR U5 sequence. The retroviral RNA sequence in the 3' region typically comprises or consists (in 5' to 3' order) of a retroviral LTR U3 sequence followed by a retroviral LTR R sequence.

[0055] The terms "DNA provirus" or "DNA provirus sequence" and "DNA proviral sequence" refer interchangeably to a DNA sequence that is integrated into the genome of a cell transduced with a retrovirus. The DNA proviral sequence contains additional regions of nucleic acid not found within the retroviral RNA sequence, including the 5' LTR U3 sequence and the 3' LTR U5 sequence. Thus, the sequences of a DNA provirus and a retroviral RNA sequence are not identical, but the sequence of a retroviral RNA sequence is shorter than the proviral DNA sequence from which it is derived. The exact 5' and 3' limits of a retroviral RNA sequence compared to the proviral DNA sequence from which it is derived cannot be easily and reliably determined by simple analysis of the proviral DNA sequence.

[0056] The retroviral vectors of the present invention comprise codon-substituted retroviral RNA sequences. Those skilled in the art will recognize that codon substitution is a technique for conferring advantageous properties to the resulting retroviral RNA sequence, such as reducing the length of the retroviral ORF and / or maximizing protein expression. For example, codon substitution includes methods for reducing the length of the retroviral ORF and, therefore, the length of any encoded retroviral (poly)peptide, and / or increasing the translation efficiency of the encoding gene. Translation efficiency can be increased by modifying the nucleic acid sequence. Codon substitution is routine in the art, and it is within the routine practice of one of skill in the art to devise a codon-substituted version of a given nucleic acid sequence. However, predicting the effect of codon substitution on other parameters is not straightforward. As a non-limiting example, as described herein, conventional wisdom teaches that codon substitution can reduce vector yield and / or transgene expression under normal manufacturing conditions.

[0057] In addition to codon substitutions, the retroviral RNA sequences of the present invention additionally contain modifications to reduce the number of retroviral open reading frames (ORFs). Those skilled in the art will recognize that an open reading frame is a span of DNA or RNA sequence between a start codon and a stop codon. ORFs can be easily identified using standard techniques known in the art, for example, by using software tools such as ORFfinder from the NIH (ORFfinder Home-NCBI (nih.gov)). Standard methods for testing the effect of ORFs, for example, on vector yield and / or transgene expression, are also within the routine skill of those skilled in the art, and exemplary methods are described herein. Retroviral ORFs are ORFs present in the (unmodified) retroviral RNA sequence that can potentially be expressed in a patient to produce retroviral proteins. Partially or completely overlapping ORFs often appear on the same nucleic acid strand. Furthermore, competing ORFs generally reside on different nucleic acid strands. After administration of a retroviral vector, expression of one or more retroviral open reading frames (ORFs) to produce retroviral proteins may theoretically trigger an immune response. Specifically, in this context, the terms "ORF reduction," "ORF elimination," and "ORF disruption" refer interchangeably to the removal of open reading frames, i.e., reducing the number of ORFs that are translated to express retroviral protein, peptide, or polypeptide sequences. This can be achieved by any suitable technique, for example, by deleting the start codon (otherwise known as the initiation codon) of the ORF. Alternatively, nucleotides in the initiation codon may be substituted, or one or more additional nucleotides may be added to disrupt the initiation codon. Those skilled in the art will further recognize that the initiation codon in a retroviral RNA sequence is AUG. The initiation codon in the corresponding proviral DNA sequence is ATG.

[0058] A stop codon signals the termination of translation. Those skilled in the art will recognize that standard stop codons in retroviral RNA sequences may be selected from UAG, UAA, and UGA. Standard stop codons in the corresponding proviral DNA sequence are TAG, TAA, and TGA.

[0059] The retroviral vectors of the present invention may additionally comprise a codon-optimized retroviral RNA sequence. Those skilled in the art will recognize that codon optimization is a technique for maximizing protein expression. For example, codon optimization can increase the translation efficiency of a coding gene. Translation efficiency can be increased by modifying the nucleic acid sequence. Codon optimization is routine in the art, and it is within the routine practice of those skilled in the art to devise a codon-optimized version of a given nucleic acid sequence. However, it is not straightforward to predict the effect of codon optimization on other parameters. As a non-limiting example, as described herein, conventional wisdom teaches that under normal production conditions, codon optimization of the gag-pol gene typically reduces vector yield.

[0060] As used herein, the terms "titer" and "yield" are used interchangeably to refer to the amount of lentiviral (e.g., SIV) vector produced by the methods of the present invention. Titer is a key benchmark for characterizing production efficiency, with a higher titer generally indicating that more retroviral / lentiviral (e.g., SIV) vectors are produced (e.g., using the same amount of reagents). Titer or yield can relate to the number of vector genomes integrated into the genome of target cells (integration titer), i.e., the number of particles capable of transducing cells, which is a measure of "active" viral particles. Transducing units (TU / mL, also referred to as TTU / mL) are a biological readout of the number of host cells transduced under certain tissue culture / virus dilution conditions and are a measure of the number of "active" viral particles. The total number of (active + inactive) viral particles may also be determined using any appropriate means, for example, by measuring either how many Gag molecules are present in the test solution or how many copies of viral RNA are in the test solution. A lentiviral particle is then assumed to contain either 2000 Gag molecules or 2 viral RNA molecules. Once the total particle number and transduction titer / TU are determined, the particle:infectivity ratio is calculated. Amino acids are referred to herein using the amino acid name, three-letter abbreviation, or one-letter abbreviation.

[0061] As used herein, the terms "protein" and "polypeptide" are used interchangeably to designate a series of amino acid residues connected to one another by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues. The terms "protein" and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycosylated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. Although "protein" and "polypeptide" are often used in reference to relatively large polypeptides, while the term "peptide" is often used in reference to small polypeptides, the usage of these terms overlaps in the art. The terms "protein" and "polypeptide" are used interchangeably herein when referring to gene products and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0062] As used herein, the terms "polynucleotide," "nucleic acid," and "nucleic acid sequence" refer to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. A nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid that is not derived from any double-stranded DNA. In one embodiment, a nucleic acid can be DNA. In another embodiment, a nucleic acid can be RNA. A suitable nucleic acid molecule is DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including siRNA, shRNA, and antisense oligonucleotides. The terms "transgene" and "gene" are also used interchangeably, and both terms encompass fragments or variants thereof that encode target proteins.

[0063] Transgenes of the present invention include nucleic acid sequences removed from their naturally occurring environment, recombinant or cloned DNA isolates, and chemically synthesized analogs or biologically synthesized analogs produced by heterologous systems.

[0064] Minor variations in the amino acid sequences of the present invention are contemplated as encompassed by the present invention, provided that the variations in the amino acid sequences maintain at least 60%, at least 70%, more preferably at least 80%, at least 85%, at least 90%, at least 95%, and most preferably at least 97% or at least 99% sequence identity with the amino acid sequences of the present invention or fragments thereof as defined elsewhere herein. The term "homology" is used herein to mean identity. Thus, variant or analog sequences of the amino acid sequences of the present invention may differ based on substitutions (typically conservative substitutions), deletions, or insertions. Proteins containing such variations are referred to herein as variants.

[0065] Proteins of the invention may include variants in which an amino acid residue from one species is substituted for the corresponding residue in another species, either at conserved or non-conserved positions. Variants of the protein molecules disclosed herein can be produced and used in the present invention. Following the lead of computational chemistry in applying multivariate data analysis techniques to structure / property-activity relationships [see, e.g., Wold, et al. Multivariate data analysis in chemistry. Chemometrics-Mathematics and Statistics in Chemistry (Ed.: B. Kowalski); D. Reidel Publishing Company, Dordrecht, Holland, 1984 (ISBN 90-277-1846-6)], quantitative activity-property relationships of proteins can be derived using well-known mathematical techniques, such as statistical regression, pattern recognition, and classification [see, e.g., Norman et al. Applied Regression Analysis. Wiley-Interscience; 3rd edition (April 1998) ISBN: 0471170828; Kandel, Abraham et al. Computer-Assisted Reasoning in Cluster Analysis. Prentice Hall PTR, (May 11, 1995), ISBN: 0133418847;Krzanowski, Wojtek. Principles of Multivariate Analysis: A User's Perspective (Oxford Statistical Science Series, No 22 (Paper)). Oxford University Press; (December 2000), ISBN: 0198507089;Witten, Ian H. et al Data Mining: Practical Machine Learning Tools and Techniques with Java Implementations.See Morgan Kaufmann; (October 11, 1999), ISBN: 1558605525; Denison David GT (Editor) et al. Bayesian Methods for Nonlinear Classification and Regression (Wiley Series in Probability and Statistics). John Wiley & Sons; (July 2002), ISBN: 0471490369; Ghose, Arup K. et al. Combinatorial Library Design and Evaluation Principles, Software, Tools, and Applications in Drug Discovery. ISBN: 0-8247-0487-8. Protein properties can be derived from empirical and theoretical models of protein sequence, function, and three-dimensional structure (e.g., analysis of potential contact residues or calculated physicochemical properties), and these properties can be considered individually and in combination.

[0066] Amino acids are referred to herein using the amino acid name, three-letter abbreviation, or one-letter abbreviation. The term "protein," as used herein, includes proteins, polypeptides, and peptides. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "protein." In some instances, the term "amino acid sequence" is synonymous with the term "peptide." The terms "protein" and "polypeptide" are used interchangeably herein. In this disclosure and claims, conventional one-letter and three-letter codes for amino acid residues may be used. The three-letter code for amino acids is as defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0067] Amino acid residues at non-conserved positions may be substituted with conserved or non-conserved residues. Conservative amino acid substitutions are particularly contemplated.

[0068] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, or histidine), acidic side chains (e.g., aspartic acid or glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, or histidine). Thus, when an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the amino acid substitution is considered conservative. The inclusion of conservatively modified variants in the proteins of the invention does not exclude other forms of variants, for example, polymorphic variants, interspecies homologs, and alleles.

[0069] "Non-conservative amino acid substitutions" include (i) substitutions of a residue having an electropositive side chain (e.g., Arg, His, or Lys) for or by an electronegative residue (e.g., Glu or Asp), (ii) substitutions of a hydrophilic residue (e.g., Ser or Thr) for or by a hydrophobic residue (e.g., Ala, Leu, Ile, Phe, or Val), (iii) substitutions of cysteine ​​or proline for or by any other residue, or (iv) substitutions of a residue having a bulky hydrophobic or aromatic side chain (e.g., Val, His, Ile, or Trp) for or by a residue having a smaller side chain (e.g., Ala or Ser) or no side chain (e.g., Gly).

[0070] "Insertions" or "deletions" are typically in the range of about 1, 2, or 3 amino acids. Acceptable variations may be determined empirically by systematically introducing amino acid insertions or deletions into a protein using recombinant DNA techniques and assaying the resulting recombinant variants for activity. This does not require more experimentation than is routine for one of ordinary skill in the art.

[0071] A "fragment" of a polypeptide comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or more of the original polypeptide.

[0072] The polynucleotides of the present invention may be prepared by any means known in the art. For example, large quantities of polynucleotides may be produced by replication in a suitable host cell. Natural or synthetic DNA fragments encoding the desired fragments are incorporated into recombinant nucleic acid constructs, typically DNA constructs, capable of being introduced into and replicated in prokaryotic or eukaryotic cells. Typically, DNA constructs are suitable for autonomous replication in unicellular hosts, such as yeast or bacteria, but may also be intended for introduction into and integration into the genome of cultured insect, mammalian, plant, or other eukaryotic cell lines.

[0073] Polynucleotides of the invention may also be produced by chemical synthesis, for example, by the phosphoramidite method or the triester method, which may be performed in a commercial automated oligonucleotide synthesizer. Double-stranded fragments may be obtained from the single-stranded product of chemical synthesis by either synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

[0074] The term "isolated" in the context of the present invention, when applied to a nucleic acid sequence, denotes that the polynucleotide sequence has been removed from its natural genetic environment, and therefore does not contain other extraneous or undesired coding sequences (but may include naturally occurring 5' and 3' untranslated regions, e.g., promoters and terminators), and is in a form suitable for use within engineered protein production systems. Such isolated molecules are those that are separated from their natural environment.

[0075] In view of the degeneracy of the genetic code, numerous sequence variations are possible among the polynucleotides of the present invention. Degenerate codons that encompass all possible codons for a given amino acid are shown below. [Table 1]

[0076] Those skilled in the art will recognize that there is flexibility in determining degenerate codons that represent all possible codons that encode each amino acid. For example, some polynucleotides encompassed by a degenerate sequence may encode variant amino acid sequences, and those skilled in the art can readily identify such variant sequences by reference to the amino acid sequences of the present invention.

[0077] A "variant" nucleic acid sequence has substantial homology or substantial similarity to a reference nucleic acid sequence (or a fragment thereof). A nucleic acid sequence or a fragment thereof is "substantially homologous" (or "substantially identical") to a reference sequence if, when optimally aligned (with appropriate nucleotide insertions or deletions) with another nucleic acid (or its complementary strand), there is nucleotide sequence identity over at least about 70%, 75%, 80%, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or more of the nucleotide bases. Methods for determining nucleic acid sequence homology are known in the art.

[0078] Alternatively, a "variant" nucleic acid sequence is substantially homologous (or substantially identical) to a reference sequence (or a fragment thereof) if the "variant" and the reference sequence can hybridize under stringent (e.g., highly stringent) hybridization conditions. Hybridization of nucleic acid sequences is affected by conditions such as salt concentration (e.g., NaCl), temperature, or organic solvents, in addition to base composition, length of complementary strands, and number of nucleotide base mismatches between hybridizing nucleic acids, as will be readily recognized by those skilled in the art. Stringent temperature conditions are preferably used and generally include temperatures above 30°C, typically above 37°C, and preferably above 45°C. Stringent salt conditions are usually less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. pH is typically between 7.0 and 8.3. The combination of parameters is much more important than any single parameter.

[0079] The method for determining nucleic acid sequence identity percentage is known in the art.For example, when evaluating nucleic acid sequence identity, a sequence having a defined number of consecutive nucleotides can be aligned with the nucleic acid sequence (having the same number of consecutive nucleotides) from the corresponding part of the nucleic acid sequence of the present invention.The tool known in the art for determining nucleic acid sequence identity percentage includes Nucleotide BLAST (described below).

[0080] Those skilled in the art will recognize that different species exhibit "preferred codon usage." As used herein, the term "preferred codon usage" refers to the codon most frequently used in cells of a particular species, thereby favoring one or a small number of possible codons encoding each amino acid. For example, the amino acid threonine (Thr) can be encoded by ACA, ACC, ACG, or ACT, but in mammalian host cells, ACC is the most commonly used codon, while in other species, different codons may be preferred. Preferred codons for a particular host cell species can be introduced into the polynucleotides of the invention by various methods known in the art. Introduction of preferred codon sequences into recombinant DNA can enhance protein production, for example, by making protein translation more efficient within a particular cell type or species. Therefore, according to the present invention, any nucleic acid sequence, in addition to the gag-pol gene, may be codon-optimized for expression in a host or target cell. In particular, the vector genome (or corresponding plasmid), the REV gene (or corresponding plasmid), the fusion protein (F) gene (or corresponding plasmid) and / or the hemagglutinin-neuraminidase (HN) gene (or corresponding plasmid), or any combination thereof may be codon optimized.

[0081] A "fragment" of a desired polynucleotide comprises a series of contiguous nucleotides from the sequence of said full-length polynucleotide. By way of example, a "fragment" of a desired polynucleotide may comprise (or consist of) at least 30 contiguous nucleotides from the sequence of said polynucleotide (e.g., at least 35, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 contiguous nucleic acid residues of said polynucleotide). A fragment may comprise at least one antigenic determinant and / or encode at least one antigenic epitope of the desired corresponding polypeptide. Typically, fragments as defined herein retain the same function as the full-length polynucleotide.

[0082] The terms "reduce," "reduced," "reduction," or "inhibit" are all used herein to mean a statistically significant amount of reduction. The terms "reduce," "reduction," or "reducing" or "inhibiting" typically mean a reduction of at least 10% compared to a reference level (e.g., in the absence of a given treatment), and can include, for example, a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, "reduction" or "inhibition" encompasses complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition (i.e., abolition) compared to a reference level.

[0083] The terms "increased," "increase," "enhance," or "activate" are all used herein to mean an increase by a statistically significant amount. The terms "increased," "increase," "enhance," or "activate" can mean an increase of at least about 25% or at least about 50% compared to a reference level, e.g., at least about 50%, or at least about 75%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 250%, or more, compared to a reference level, or an increase of at least about 1.5-fold, or at least about 2-fold, or at least about 2.5-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or more, compared to a reference level. In the context of yield or titer, an "increase" is an observable or statistically significant increase in such level.

[0084] The terms "individual," "subject," and "patient" are used interchangeably herein to refer to a mammalian subject for whom diagnosis, prognosis, disease monitoring, treatment, therapy, and / or therapy optimization is desired. The mammal may be (but is not limited to) a human, non-human primate, mouse, rat, dog, cat, horse, or cow. In preferred embodiments, the individual, subject, or patient is human. An "individual" may be an adult, juvenile, or infant. An "individual" may be male or female.

[0085] A "subject in need" of treatment for a particular condition can be an individual who has the condition, has been diagnosed with the condition, or is at risk of developing the condition.

[0086] The subject may be a subject who has been previously diagnosed or identified as suffering from or having a condition in need of treatment, or one or more complications or symptoms associated with such a condition, and may have already received treatment for a condition as defined herein or one or more complications or symptoms associated with said condition. Alternatively, the subject may also be a subject who has not previously been diagnosed with a condition as defined herein or one or more symptoms or complications associated with said condition. For example, the subject may be a subject who exhibits one or more risk factors for a condition, or one or more symptoms or complications associated with said condition, or a subject who does not exhibit risk factors.

[0087] As used herein, the term "healthy individual" refers to an individual or group of individuals in a healthy state, e.g., individuals who do not exhibit any symptoms of a disease, have not been diagnosed with a disease, and / or are unlikely to develop a disease, e.g., cystic fibrosis (CF) or any other disease described herein. Preferably, the healthy individual is not receiving medication that affects CF and has not been diagnosed with any other disease. One or more healthy individuals may have similar gender, age, and / or body mass index (BMI) compared to the test individual. Application of standard statistical methods used in medicine allows for the determination of normal levels of expression in healthy individuals, and significant deviations from such normal levels.

[0088] As used herein, the terms "control" and "reference population" are used interchangeably.

[0089] The term "pharmaceutically acceptable," as used herein, means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopeia.

[0090] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application, and should not be construed herein as an admission that such publications constitute prior art to the claims appended hereto.

[0091] Disclosure relating to the various methods of the invention is intended to apply equally to other methods, therapeutic uses or methods, data storage media or devices, computer program products, and vice versa.

[0092] Retroviral and lentiviral vectors The present invention relates to retroviral / lentiviral (e.g., SIV) vectors. The term "retrovirus" refers to any member of the Retroviridae family of RNA viruses that encode the enzyme reverse transcriptase. The term "lentivirus" refers to the family of retroviruses. Examples of suitable retroviruses for use in the present invention include gammaretroviruses, such as murine leukemia virus (MLV) and feline leukemia virus (FLV). Examples of suitable lentiviruses for use in the present invention include simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), and visna / maedi virus. Preferably, the present invention relates to lentiviral vectors and their generation. Particularly preferred lentiviral vectors are SIV vectors (including all strains and subtypes), such as SIV-AGM (originally isolated from the African green monkey, Cercopithecus aethiops). Alternatively, the present invention relates to HIV vectors.

[0093] The retroviral / lentiviral (e.g., SIV) vectors of the invention are typically pseudotyped with the hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus, preferably Sendai virus (murine parainfluenza virus type 1).

[0094] The F protein may be a truncated F protein, typically one in which the cytoplasmic domain is truncated. Preferably, the truncated F protein is Fct4 in which 38 amino acids have been truncated from the C-terminus of the F protein and 4 amino acids of the F protein cytoplasmic domain have been retained. Thus, the F protein may comprise or consist of an Fct4 amino acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100%, identity to SEQ ID NO: 12 or 13. Preferably, the F protein may comprise or consist of an Fct4 amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 12 or 13.

[0095] The full-length F protein, or a C-terminally truncated form thereof (e.g., Fct4), is typically fusion-inactive. The fusion-inactive form of the F protein may be cleaved to generate two subunits: a first subunit (also known as F2) and a second subunit (also known as F1).

[0096] The first subunit of the F protein may comprise or consist of an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100%, identity to SEQ ID NO: 14. Preferably, the first subunit may be a subunit that comprises or consists of an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 14. SEQ ID NO: 14 is the first subunit of Fct4.

[0097] Alternatively or additionally, and preferably additionally, the second subunit of the F protein may comprise or consist of an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100%, identity to SEQ ID NO: 15. Preferably, the second subunit may be a subunit that comprises or consists of an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 15. SEQ ID NO: 15 is the second subunit of Fct4.

[0098] An F protein (e.g., Fct4) may contain an N-terminal signal peptide. Alternatively, the F protein may lack such a signal peptide. The F protein signal peptide may comprise or consist of an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100%, identity to SEQ ID NO: 16. This signal peptide may be cleaved to form the mature F protein. The signal peptide of Fct4 is SEQ ID NO: 16, which forms amino acid residues 1-25 of SEQ ID NO: 13. Thus, the mature form of Fct4 may comprise or consist of an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or more, up to 100%, identity to amino acid residues 26 to 527 of SEQ ID NO: 13.

[0099] Within pGM301, an exemplary F protein plasmid (pDNA3a), there are potential alternative start codons upstream of the start codon that initiates translation to produce Fct4 of SEQ ID NOS: 12 and 13. However, according to the present invention, the F proteins of retroviral / lentiviral (e.g., SIV) vectors of the invention do not include additional amino acid sequences N-terminal to the methionine at position 1 in SEQ ID NO: 13. In particular, the F proteins of retroviral / lentiviral (e.g., SIV) vectors of the invention typically do not include one or more amino acids corresponding to those encoded by bases 1645-1734 of pGM301 (SEQ ID NO: 23), which is translated as MFMPSSFSYSSWATCWLLCCLIILAKNSIA (SEQ ID NO: 46) N-terminal to the methionine at position 1 in SEQ ID NO: 13.

[0100] The HN protein may be a truncated and / or chimeric HN protein, typically one in which the cytoplasmic domain is truncated or replaced. Preferably, the HN protein is a chimeric HN protein in which (i) the cytoplasmic domain of HN is replaced by the cytoplasmic domain of a transmembrane protein (TMP); or (ii) the cytoplasmic domain of a TMP is added to the cytoplasmic domain of the HN protein. The HN protein may be as described in Kobayashi et al. (J. Virol. (2003) 77(4):2607-2614), which is incorporated herein by reference in its entirety.

[0101] F / HN pseudotyping is particularly effective for targeting cells of airway epithelium, and therefore for therapeutic use, it is typically delivered to cells of the respiratory tract, including cells of airway epithelium.Therefore, the retroviral / lentiviral (e.g., SIV) vector of the present invention is particularly suitable for treating diseases or disorders of the respiratory tract, respiratory airway, or lungs.Typically, the retroviral / lentiviral (e.g., SIV) vector can be used to treat genetic respiratory diseases.

[0102] Retroviral / lentiviral (e.g., SIV) vectors of the invention may be pseudotyped with proteins from another virus, provided that the combination of modified retroviral / lentiviral (e.g., SIV) RNA sequences and / or the use of codon-optimized gag-pol genes (e.g., from SIV) does not negatively impact the vector's production titer (or even result in increased vector titer) and / or transgene expression (or even result in increased transgene expression). Non-limiting examples of other proteins that can be used to pseudotype retroviral / lentiviral (e.g., SIV) vectors of the invention include the G glycoprotein from vesicular stomatitis virus (G-VSV) and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, or modified forms thereof; for example, those described in UK Patent Applications Nos. 2118685.3 and 2105278.2, each of which is incorporated herein by reference in its entirety.

[0103] The retroviral / lentiviral (e.g., SIV) vector of the present invention further comprises Gag, Pol, and / or GagPol. Typically, the Gag, Pol, and / or GagPol are derived from the desired retroviral / lentiviral (e.g., SIV) vector. As a non-limiting example, when the retroviral vector of the present invention is SIV, the Gag, Pol, and / or GagPol are typically derived from SIV.

[0104] The Gag, Pol, and / or GagPol sequences may be codon-optimized. The present inventors have previously shown that, unexpectedly, the production titer of retroviral vectors containing codon-optimized Gag, Pol, and / or GagPol polyproteins derived from SIV is not negatively affected (see International Application No. PCT / GB2022 / 050524, which is incorporated herein by reference in its entirety). Indeed, the present inventors have previously shown that the production titer of retroviral vectors pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins derived from respiratory paramyxoviruses and containing codon-optimized Gag, Pol, and / or GagPol derived from SIV can be further increased. This benefit of maintained / improved retroviral / lentiviral (e.g., SIV) vector yield can be combined with the benefits of the present invention in terms of providing retroviral / lentiviral (e.g., SIV) vectors with maintained / increased transgene expression and / or maintained / increased retroviral / lentiviral (e.g., SIV) RNA sequence integration, while addressing potential safety risks and improving the safety profile of the retroviral / lentiviral (e.g., SIV) vectors described herein.

[0105] In the context of Gag, Pol, and / or GagPol, codon optimization is a technique for maximizing protein expression by increasing the translation efficiency of the encoding gene. Translation efficiency is increased by modifying the nucleic acid sequence. Codon optimization is routine in the art, and it is within the routine practice of one of ordinary skill in the art to devise a codon-optimized version of a given nucleic acid sequence. However, what is not straightforward is predicting the effect of codon optimization on other parameters. For example, as described herein, conventional wisdom teaches that under normal production conditions (when the vector genome plasmid is limiting, rather than the gag-pol gene), codon optimization of the gag-pol gene typically reduces vector yield.

[0106] Retroviral / lentiviral (e.g., SIV) vectors of the invention may comprise a codon-optimized Gag protein, a codon-optimized Pol protein, a codon-optimized GagPol polyprotein, or a combination thereof. Accordingly, the present invention provides retroviral / lentiviral (e.g., SIV) vectors comprising a codon-optimized Gag protein comprising, or consisting of, an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100%, sequence identity to SEQ ID NO:9. Preferably, the present invention provides retroviral vectors comprising a codon-optimized Gag protein comprising, or consisting of, an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:9. The present invention provides retroviral vectors comprising a codon-optimized Pol protein comprising, or consisting of, an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100%, sequence identity to SEQ ID NO: 10. Preferably, the present invention provides retroviral vectors comprising a codon-optimized Pol protein comprising, or consisting of, an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 10.

[0107] GagPol is expressed as a polyprotein that is processed within the viral particle to generate several smaller proteins. The extent of processing, and therefore the presence and / or concentration of GagPol or any of the component proteins within the retroviral / lentiviral (e.g., SIV) vectors of the invention, may vary over time.

[0108] Thus, retroviral / lentiviral (e.g., SIV) vectors of the present invention may comprise one or more of p17 protein, p27 protein, p8 protein, protease, p51 protein, p15 protein, and p31 protein. One or more of these proteins may be present in combination with Gag, Pol, and / or GagPol. Preferably, the present invention provides retroviral vectors comprising p17 protein, p27 protein, p8 protein, protease, p51 protein, p15 protein, and p31 protein. These proteins may also be present in combination with Gag, Pol, and / or GagPol.

[0109] The p17 protein may comprise or consist of an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or more, up to 100%, sequence identity to SEQ ID NO: 2. Preferably, the p17 protein comprises or consists of an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 2.

[0110] The p24 protein may comprise or consist of an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100%, sequence identity to SEQ ID NO: 3. Preferably, the p24 protein comprises or consists of an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 3.

[0111] The p8 protein may comprise or consist of an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or more, up to 100%, sequence identity to SEQ ID NO: 4. Preferably, the p8 protein comprises or consists of an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:4.

[0112] The protease may comprise or consist of an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or more, up to 100% sequence identity to SEQ ID NO: 5. Preferably, the protease comprises or consists of an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:5.

[0113] The p51 protein may comprise or consist of an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or more, up to 100%, sequence identity to SEQ ID NO: 6. Preferably, the p51 protein comprises or consists of an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 6.

[0114] The p15 protein may comprise or consist of an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or more, up to 100%, sequence identity to SEQ ID NO: 7. Preferably, the p15 protein comprises or consists of an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 7.

[0115] The p31 protein may comprise or consist of an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100%, sequence identity to SEQ ID NO: 8. Preferably, the p31 protein comprises or consists of an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:8.

[0116] Retroviral / lentiviral (e.g., SIV) vectors of the invention include a p17 protein (as described above) that comprises or consists of an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity to SEQ ID NO:2, at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity to SEQ ID NO:3, p24 protein (as described above) comprising or consisting of an amino acid sequence having up to 100% sequence identity to SEQ ID NO:4; p8 protein (as described above) comprising or consisting of an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or up to 100% sequence identity to SEQ ID NO:5; a protease (as described above) comprising or consisting of an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity to SEQ ID NO:6; a p51 protein (as described above) comprising or consisting of an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity to SEQ ID NO:7; 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity to SEQ ID NO:8; and p31 protein (as described above) comprising or consisting of an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity to SEQ ID NO:8.

[0117] Retroviral / lentiviral (e.g., SIV) vectors according to the present invention may be integrase-competent (IC), or alternatively, retroviral / lentiviral (e.g., SIV) vectors may be integrase-deficient (ID).

[0118] Retroviral / lentiviral (e.g., SIV) vectors, such as those of the present invention, can integrate into the genome of transduced cells and result in long-lasting expression, making them suitable for transduction of stem / progenitor cells. In the lung, several cell types with regenerative capacity have been identified as responsible for maintaining specific cell lineages in the conducting airways and alveoli. These include basal cells and submucosal gland duct cells in the upper respiratory tract, club cells and neuroendocrine cells in the bronchiolar airways, bronchoalveolar epithelial stem cells in the terminal bronchioles, and type II pneumocytes in the alveoli. Therefore, without being bound by theory, it is believed that the retroviral / lentiviral (e.g., SIV) vectors result in long-term gene expression of a desired transgene by introducing the transgene into one or more long-lived airway epithelial cells or cell types, such as basal cells and submucosal gland duct cells in the upper respiratory tract, club cells and neuroendocrine cells in the bronchiolar airways, bronchoalveolar epithelial stem cells in the terminal bronchioles, and type II pneumocytes in the alveoli. As demonstrated herein, integration of retroviral / lentiviral (e.g., SIV) vectors carrying the modified retroviral / lentiviral (e.g., SIV) RNA sequences of the present invention into the target cell genome is unexpectedly not negatively affected and, in fact, can even be increased.

[0119] Thus, the retroviral / lentiviral (e.g., SIV) vectors of the present invention can be used to transduce one or more cells or cell lines with regenerative potential within the lungs (including the airways and respiratory tract) to achieve long-term gene expression. For example, retroviral / lentiviral (e.g., SIV) vectors can transduce basal cells, such as those in the upper / respiratory tract. Basal cells play a central role in the process of epithelial maintenance and repair after injury. In addition, basal cells are widely distributed along the human respiratory epithelium, with a relative distribution ranging from 30% (large airways) to 6% (small airways).

[0120] The retroviral / lentiviral (e.g., SIV) vectors of the invention may be used to transduce isolated and expanded stem / progenitor cells ex vivo prior to administration to a patient. Preferably, the retroviral / lentiviral (e.g., SIV) vectors of the invention are used to transduce cells in the lungs (or airways / respiratory tract) in vivo.

[0121] The retroviral / lentiviral (e.g., SIV) vectors of the present invention demonstrate remarkable resistance to shear forces when passed through clinically relevant delivery devices, such as bronchoscopes, spray bottles, and nebulizers, with only a modest reduction in transduction capacity.

[0122] The retroviral / lentiviral (e.g., SIV) vectors of the present invention enable high levels of transgene expression, resulting in high levels (therapeutic levels) of expression of therapeutic proteins. The retroviral / lentiviral (e.g., SIV) vectors of the present invention typically provide high levels of transgene expression when administered to a patient. The terms high expression and therapeutic expression are used interchangeably herein. Expression may be measured by any appropriate method (qualitative or quantitative, preferably quantitative) and by any appropriate unit of measurement, e.g., concentration given in ng / ml or nM.

[0123] Expression of a desired transgene may be given relative to the expression of the corresponding endogenous (defective) gene in the patient. Expression may be measured in terms of mRNA or protein expression. Expression of a transgene of the invention, e.g., a functional CFTR gene, may be quantified relative to the endogenous gene, e.g., an endogenous (dysfunctional) CFTR gene, in terms of mRNA copies per cell or any other suitable unit.

[0124] The expression level of the transgene and / or encoded therapeutic protein of the invention may be measured in lung tissue, epithelial lining fluid, and / or serum / plasma, as appropriate. High and / or therapeutic expression levels may therefore refer to concentrations in the lung, epithelial lining fluid, and / or serum / plasma.

[0125] The retroviral / lentiviral (e.g., SIV) vectors of the present invention exhibit efficient airway cell uptake, enhanced transgene expression, and do not suffer from the drawback of loss of efficacy upon repeated administration. Thus, the retroviral / lentiviral (e.g., SIV) vectors of the present invention can produce long-lasting, reproducible, and high-level expression in airway cells without inducing excessive immune responses.

[0126] The retroviral / lentiviral (e.g., SIV) vectors of the present invention enable long-term transgene expression, resulting in long-term expression of therapeutic proteins. As used herein, the terms "long-term expression," "sustained expression," "long-lasting expression," and "persistent expression" are used interchangeably. Long-term expression according to the present invention refers to expression of a therapeutic gene and / or protein, preferably at therapeutic levels, for at least 45 days, at least 60 days, at least 90 days, at least 120 days, at least 180 days, at least 250 days, at least 360 days, at least 450 days, at least 730 days, or more. Preferably, long-term expression refers to expression for at least 90 days, at least 120 days, at least 180 days, at least 250 days, at least 360 days, at least 450 days, at least 720 days, or more, more preferably at least 360 days, at least 450 days, at least 720 days, or more. This long-term expression can be achieved by repeated doses or by a single dose.

[0127] Repeat doses may be administered twice daily, daily, twice weekly, weekly, monthly, every two months, every three months, every four months, every six months, yearly, every two years, or more. Dosing may be continued for as long as needed, for example, for at least six months, at least one year, two years, three years, four years, five years, ten years, fifteen years, twenty years, or more, up to the lifespan of the patient being treated.

[0128] Preferably, the present invention relates to F / HN retroviral / lentiviral vectors, in particular SIV F / HN vectors, comprising a promoter and a transgene.

[0129] Retroviral and lentiviral RNA sequences Each retroviral vector particle contains a retroviral RNA sequence. The retroviral RNA sequence, together with a transgene expression cassette, contains an LTR element, a sequence necessary for incorporation into the particle. As a non-limiting example, the retroviral RNA sequence, together with the transgene expression cassette, may contain or consist of a retroviral LTR element, a retroviral sequence necessary for incorporation into the retroviral particle (typically R and U5 at the 5' end of the sequence (reading 5' to 3') and U3 and R at the 3' end of the sequence (reading 5' to 3')). The transgene expression cassette typically consists of a suitable enhancer / promoter element, transgene cDNA, and post-transcriptional regulatory elements. Particularly preferred is a retroviral RNA sequence that, together with the transgene expression cassette, contains an SIV LTR element, a sequence necessary for incorporation into the particle. As a non-limiting example, the SIV RNA sequence, together with the transgene expression cassette, may comprise or consist of SIV LTR elements (typically R and U5 at the 5' end of the sequence (reading 5' to 3') and U3 and R at the 3' end of the sequence (reading 5' to 3')), which are SIV sequences necessary for incorporation into retroviral particles.

[0130] The retroviral or lentiviral RNA sequences of the present invention are modified compared to the unmodified retroviral or lentiviral RNA sequence from which they are derived. Modification of the retroviral or lentiviral RNA sequence may provide advantageous properties compared to the retroviral or lentiviral RNA sequence from which it is derived. Non-limiting examples of such advantageous properties include maintained / increased transgene expression, maintained / increased retroviral / lentiviral (e.g., SIV) RNA sequence integration into the target / host cell genome, maintained / increased vector yield, and / or improved patient safety compared to the unmodified retroviral or lentiviral RNA sequence from which it is derived.

[0131] The modified retroviral or lentiviral RNA sequence of the present invention may have codon substitutions and / or contain a reduced number of retroviral or lentiviral ORFs compared to the retroviral or lentiviral RNA sequence from which it is derived. For example, the modified retroviral or lentiviral RNA sequence of the present invention may contain a reduced number of retroviral or lentiviral ORFs compared to the retroviral or lentiviral RNA sequence from which it is derived. Typically, the modified retroviral or lentiviral RNA sequence of the present invention has codon substitutions and contains a reduced number of retroviral or lentiviral ORFs compared to the retroviral or lentiviral RNA sequence from which it is derived.

[0132] Codon substitutions in retroviral or lentiviral RNA sequences may include, for example, the introduction of a stop codon and / or the introduction and / or removal of a restriction enzyme cleavage site. At least one, at least two, at least three, at least four, at least five, or more codons may be substituted in the modified retroviral or lentiviral genome of the present invention. For each substituted codon, the nature of the modification may be independently selected, for example, the introduction of a stop codon and / or the introduction and / or removal of a restriction enzyme cleavage site. Standard techniques for codon substitution in retroviral or lentiviral RNA sequences in this manner are known in the art. Preferably, the modified retroviral / lentiviral (e.g., SIV) RNA sequence includes one or more codon substitutions to introduce a stop codon. The introduction of a stop codon may include the introduction of a frameshift.

[0133] Introduction of a stop codon can result in premature termination of translation, resulting in an ORF of reduced length compared to the corresponding unmodified ORF in which the stop sequence has not been introduced. Therefore, according to the present invention, a retroviral or lentiviral RNA sequence is typically modified to introduce one or more stop codons, thus reducing the length of one or more ORFs. For example, the length of one or more ORFs may be reduced by introducing a UAG, UAA, or UGA codon in the retroviral RNA sequence (or a TAG, TAA, or TGA codon in the proretroviral DNA sequence). As described herein, a stop codon may be removed by deleting or substituting nucleotides in the retroviral RNA sequence or the corresponding proretroviral DNA sequence to provide a stop codon, or by adding one or more (e.g., one, two, or three) nucleotides to introduce the stop codon. Preferably, the retroviral or lentiviral RNA sequence is modified to reduce the length of one or more retroviral or lentiviral ORFs. Reducing the length of one or more retroviral or lentiviral ORFs has the potential to improve the safety of retroviral or lentiviral vectors when administered to a subject. Thus, retroviral or lentiviral vectors of the present invention comprising modified retroviral or lentiviral RNA sequences may have an improved safety profile compared to retroviral or lentiviral vectors comprising the unmodified retroviral or lentiviral RNA sequence from which the modified retroviral or lentiviral RNA sequence is derived. As a non-limiting example, reducing the length of one or more retroviral or lentiviral ORFs reduces the risk of an immune response elicited by expression of a longer polypeptide encoded by the corresponding unmodified retroviral or lentiviral ORF(s).Additionally, as demonstrated herein, the length of one or more retroviral or lentiviral ORFs can be reduced without negatively affecting downstream transgene expression, retroviral or lentiviral vector integration, and / or retroviral or lentiviral vector yield. Reducing the length of one or more retroviral or lentiviral ORFs can increase downstream transgene expression, retroviral or lentiviral vector integration, and / or retroviral or lentiviral vector yield.

[0134] As exemplified herein, such modifications may include or consist of modifying a retroviral or lentiviral RNA sequence to introduce a stop codon to reduce the length of one or more viral, particularly retroviral / lentiviral (e.g., SIV) ORFs in said sequence compared to the unmodified retroviral or lentiviral RNA sequence from which the modified retroviral or lentiviral RNA sequence is derived. Modification of the retroviral or lentiviral RNA sequence may be achieved by modifying the vector genome plasmid (i.e., pDNA1) described herein used to generate the modified retroviral or lentiviral vector of the invention. Thus, the modified vector genome plasmid (i.e., pDNA1) may contain one or more ORFs, particularly one or more retroviral / lentiviral (e.g., SIV) ORFs, of reduced length compared to the corresponding unmodified plasmid genome vector (i.e., pDNA1).

[0135] As a non-limiting example, modified retroviral or lentiviral (e.g., SIV) RNA sequences of the invention may be modified to introduce at least one, at least two, at least three, at least four, at least five, or more stop codons, each of which typically reduces the length of a retroviral or lentiviral (e.g., SIV) ORF. Typically, the length of one or more retroviral or lentiviral (e.g., SIV) ORFs is reduced compared to the corresponding retroviral or lentiviral (e.g., SIV) ORF in the unmodified retroviral or lentiviral (e.g., SIV) RNA sequence from which the modified retroviral or lentiviral (e.g., SIV) RNA sequence is derived. Thus, vector genome plasmids used to generate modified retroviral or lentiviral (e.g., SIV) vectors of the invention may contain one or more ORFs, particularly one or more retroviral / lentiviral (e.g., SIV) ORFs, of reduced length compared to the corresponding unmodified plasmid genome vector (i.e., pDNA1).

[0136] A retroviral or lentiviral (e.g., SIV) RNA sequence may be modified to reduce the length of one or more retroviral or lentiviral (e.g., SIV) ORFs 5' (also referred to as upstream) of the transgene and / or transgene promoter. One or more retroviral or lentiviral (e.g., SIV) ORFs 5' of the transgene and / or transgene promoter may be reduced in length. As a non-limiting example, at least one, at least two, at least three, at least four, at least five, or more retroviral or lentiviral (e.g., SIV) ORFs 5' of the transgene and / or transgene promoter may be reduced in length. Preferably, one or two retroviral or lentiviral (e.g., SIV) ORFs 5' of the transgene promoter are reduced in length. The length of one or more upstream ORFs may be reduced compared to the length of the corresponding ORF in the unmodified retroviral or lentiviral (e.g., SIV) RNA sequence from which the modified retroviral or lentiviral (e.g., SIV) RNA sequence is derived. Thus, the vector genome plasmid used to generate the modified retroviral or lentiviral (e.g., SIV) vectors of the present invention may contain one or more upstream ORFs, particularly one or more upstream retroviral / lentiviral (e.g., SIV) ORFs, of a reduced length compared to the corresponding unmodified plasmid genome vector (i.e., pDNA1).

[0137] Introduction of a stop codon may reduce the length of a polypeptide encoded by a retroviral or lentiviral (e.g., SIV) ORF by at least 5 amino acids, at least 10 amino acids, at least 20 amino acids, at least 40 amino acids, or more.

[0138] Alternatively, or in addition, each introduced stop codon may reduce the length of one or more retroviral or lentiviral (e.g., SIV) ORFs encoding a polypeptide by at least 10 amino acids, e.g., at least 50 amino acids, at least 100 amino acids, at least 200 amino acids, or more, compared to the length of the unmodified ORF before the introduction of the stop codon. For example, the introduction of a stop codon may reduce the length of one or more retroviral or lentiviral (e.g., SIV) ORFs encoding a polypeptide at least 230 amino acids in length.

[0139] Thus, as a non-limiting example, the introduction of a stop codon may reduce the length of a polypeptide encoded by a retroviral or lentiviral (e.g., SIV) ORF, where (i) the polypeptide encoded by the (unmodified ORF) is at least 230 amino acids in length; and (ii) the length of the polypeptide encoded by the ORF is reduced by at least 40 amino acids or more.

[0140] The introduction of individual stop codons may reduce the length of two or more ORFs, in particular one or more retroviral / lentiviral ORFs. In particular, the introduction of individual stop codons may reduce the length of two or three ORFs, in particular two or three retroviral / lentiviral ORFs, with a reduction in the length of two ORFs being preferred.

[0141] Other codon substitutions include the removal and / or replacement of one or more restriction enzyme sites. Such codon substitutions may be useful in generating retroviral / lentiviral vectors of the invention.

[0142] Preferred codon substitutions may include or consist of frameshift mutations and stop codon replacements in the Env ORF of the retroviral / lentiviral RNA sequence. Such substitutions typically reduce the length of the Env ORF and prevent readthrough from the Env ORF to the cPPT sequence. As illustrated, one such preferred codon substitution involves replacing the motif corresponding to residues 2347-2352 of SEQ ID NO:25 with the motif corresponding to residues 2354-2360 of SEQ ID NO:19. This reduces the length of the polypeptide encoded by the Env ORF from 235 amino acids to 192 amino acids and also reduces the length of the polypeptide encoded by the additional retroviral / lentiviral ORF from 19 amino acids to 9 amino acids. The motif corresponding to residues 2354-2360 of SEQ ID NO:19 is found at residues 1601-1607 of SEQ ID NO:1.

[0143] Another preferred codon substitution that can be used instead of or in addition to the codon substitutions in the preceding paragraph is the introduction of an Sbfl restriction site, which may optionally replace an EcoRl restriction site within the retroviral / lentiviral RNA sequence. As exemplified, one such preferred codon substitution involves replacing the motif corresponding to residues 1734-1739 of SEQ ID NO:25 with the motif corresponding to residues 1738-1746 of SEQ ID NO:19. The motif corresponding to residues 1738-1746 of SEQ ID NO:19 is found at residues 985-993 of SEQ ID NO:1.

[0144] Particularly preferred are codon substitutions that comprise or consist of a combination of (a) a frameshift mutation and replacement of a stop codon into the Env ORF of the retroviral / lentiviral RNA sequence; and (b) the introduction of an Sbfl restriction site, which may optionally replace an EcoRl restriction site within the retroviral / lentiviral RNA sequence. As exemplified, particularly preferred codon substitutions comprise or consist of (a) the replacement of the motif corresponding to residues 2347-2352 of SEQ ID NO:25 with the motif corresponding to residues 2354-2360 of SEQ ID NO:25; and (b) the replacement of the motif corresponding to residues 1734-1739 of SEQ ID NO:25 with the motif corresponding to residues 1738-1746 of SEQ ID NO:25.

[0145] Retroviral or lentiviral RNA sequences are typically modified to reduce the number of ORFs. For example, the number of ORFs may be reduced by removing an AUG codon in the retroviral RNA sequence (or an ATG codon in a proretroviral DNA sequence). As described herein, the start codon may be removed by deleting or substituting nucleotides within the start codon or by adding one or more (e.g., one, two, or three) nucleotides to disrupt the start codon. Preferably, the retroviral or lentiviral RNA sequence is modified to reduce the number of retroviral or lentiviral ORFs. Removal of one or more retroviral or lentiviral ORFs has the potential to improve the safety of the retroviral or lentiviral vector when administered to a subject. Therefore, retroviral or lentiviral vectors of the present invention comprising modified retroviral or lentiviral RNA sequences may have an improved safety profile compared to retroviral or lentiviral vectors comprising the unmodified retroviral or lentiviral RNA sequence from which the modified retroviral or lentiviral RNA sequence is derived. As a non-limiting example, removal of one or more retroviral or lentiviral ORFs reduces the risk of an immune response elicited by expression of the one or more retroviral or lentiviral ORFs. Additionally, as demonstrated herein, removal of one or more retroviral or lentiviral ORFs can be achieved without negatively affecting downstream transgene expression, retroviral or lentiviral vector integration, and / or retroviral or lentiviral vector yield. Removal of one or more retroviral or lentiviral ORFs can increase downstream transgene expression, retroviral or lentiviral vector integration, and / or retroviral or lentiviral vector yield.

[0146] As exemplified herein, such modifications may include or consist of modifying a retroviral or lentiviral RNA sequence to remove viral, particularly retroviral / lentiviral (e.g., SIV) ORFs from said sequence, compared to the unmodified retroviral or lentiviral RNA sequence from which the modified retroviral or lentiviral RNA sequence is derived. Modification of the retroviral or lentiviral RNA sequence may be achieved by modifying the vector genome plasmid (i.e., pDNA1) described herein used to generate the modified retroviral or lentiviral vectors of the invention. Thus, the modified vector genome plasmid (i.e., pDNA1) may contain a reduced number of viral, particularly retroviral / lentiviral (e.g., SIV) ORFs, compared to the corresponding unmodified plasmid genome vector (i.e., pDNA1). Thus, the modified retroviral or lentiviral vectors of the invention contain a reduced number of non-transgene ORFs on their retroviral or lentiviral RNA sequence.

[0147] As a non-limiting example, modified retroviral or lentiviral (e.g., SIV) RNA sequences of the invention may be modified to remove at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more retroviral or lentiviral (e.g., SIV) ORFs, typically at least six or at least seven retroviral or lentiviral (e.g., SIV) ORFs, preferably six or seven retroviral or lentiviral (e.g., SIV) ORFs. Typically, the number of retroviral or lentiviral (e.g., SIV) ORFs is reduced compared to the unmodified retroviral or lentiviral (e.g., SIV) RNA sequence from which the modified retroviral or lentiviral (e.g., SIV) RNA sequence is derived. Thus, vector genome plasmids used to generate modified retroviral or lentiviral (e.g., SIV) vectors of the invention may have a reduced number of retroviral or lentiviral (e.g., SIV) ORFs compared to the corresponding unmodified vector genome plasmid.

[0148] The retroviral or lentiviral (e.g., SIV) RNA sequence may be modified to reduce the number of retroviral or lentiviral (e.g., SIV) ORFs 5' (also referred to as upstream) of the transgene and / or transgene promoter. One or more retroviral or lentiviral (e.g., SIV) ORFs may be removed from 5' of the transgene and / or transgene promoter. By way of non-limiting example, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more retroviral or lentiviral (e.g., SIV) ORFs may be removed from 5' of the transgene and / or transgene promoter, typically at least six or at least seven retroviral or lentiviral (e.g., SIV) ORFs, preferably six or seven retroviral or lentiviral (e.g., SIV) ORFs. Preferably, one or more retroviral or lentiviral (e.g., SIV) ORFs are removed from 5' of the transgene promoter. The number of upstream ORFs may be reduced compared to the unmodified retroviral or lentiviral (e.g., SIV) RNA sequence from which the modified retroviral or lentiviral (e.g., SIV) RNA sequence is derived. Thus, the vector genome plasmids used to generate modified retroviral or lentiviral (e.g., SIV) vectors of the invention may have a reduced number of upstream retroviral or lentiviral (e.g., SIV) ORFs compared to the corresponding unmodified vector genome plasmid.

[0149] Alternatively, or in addition, one or more retroviral or lentiviral (e.g., SIV) ORFs to be deleted in accordance with the present invention may each independently encode a polypeptide that is greater than or equal to 10 amino acids in length, greater than or equal to 20 amino acids in length, greater than or equal to 30 amino acids in length, greater than or equal to 40 amino acids in length, greater than or equal to 50 amino acids in length, greater than or equal to 60 amino acids in length, greater than or equal to 70 amino acids in length, greater than or equal to 80 amino acids in length, greater than or equal to 90 amino acids in length, greater than or equal to 100 amino acids in length, greater than or equal to 110 amino acids in length, greater than or equal to 120 amino acids in length, greater than or equal to 130 amino acids in length, greater than or equal to 140 amino acids in length, or greater than or equal to 150 amino acids in length. Typically, one or more retroviral or lentiviral (e.g., SIV) ORFs to be deleted in accordance with the present invention may each independently encode a polypeptide that is greater than or equal to 100 amino acids in length. Preferably, at least one retroviral or lentiviral (e.g., SIV) ORF encoding a polypeptide greater than or equal to 100 amino acids in length may be removed from the modified retroviral or lentiviral (e.g., SIV) RNA sequence compared to the unmodified retroviral or lentiviral (e.g., SIV) RNA sequence from which the modified retroviral or lentiviral (e.g., SIV) RNA sequence is derived. Thus, the vector genome plasmid used to generate the modified retroviral or lentiviral (e.g., SIV) vector of the present invention may have one or more retroviral or lentiviral (e.g., SIV) ORFs encoding a polypeptide greater than or equal to 100 amino acids in length removed compared to the unmodified plasmid genome vector from which the modified retroviral RNA sequence is derived.

[0150] Thus, the retroviral or lentiviral (e.g., SIV) RNA sequences of the invention may lack any ORFs (other than the transgene) that encode polypeptides greater than or equal to 200 amino acids in length, greater than or equal to 190 amino acids in length, greater than or equal to 180 amino acids in length, greater than or equal to 170 amino acids in length, or greater than or equal to 160 amino acids in length, compared to the unmodified retroviral or lentiviral (e.g., SIV) RNA sequence from which the modified retroviral or lentiviral (e.g., SIV) RNA sequence is derived. Thus, the vector genome plasmid used to generate the modified retroviral or lentiviral (e.g., SIV) vector of the invention may lack any ORFs (other than the transgene) that encode polypeptides greater than or equal to 200 amino acids in length, as described above, compared to the unmodified plasmid genome vector from which the modified retroviral or lentiviral (e.g., SIV) RNA sequence is derived.

[0151] The retroviral or lentiviral (e.g., SIV) RNA sequences of the present invention may lack any ORFs encoding polypeptides greater than or equal to 180 amino acids in length, greater than or equal to 100 amino acids in length, greater than or equal to 90 amino acids in length, greater than or equal to 80 amino acids in length, or greater than or equal to 70 amino acids in length within the partial Gag region, compared to the unmodified retroviral or lentiviral (e.g., SIV) RNA sequence from which the modified retroviral or lentiviral (e.g., SIV) RNA sequence is derived. Thus, the vector genome plasmid used to generate the modified retroviral or lentiviral (e.g., SIV) vectors of the present invention may lack any ORFs (other than a transgene) encoding polypeptides greater than or equal to 180 amino acids in length within the partial Gag region described above, compared to the unmodified plasmid genome vector from which the modified retroviral or lentiviral (e.g., SIV) RNA sequence is derived.

[0152] The retroviral or lentiviral (e.g., SIV) RNA sequences of the present invention may lack any ORFs encoding polypeptides greater than or equal to 200 amino acids in length, greater than or equal to 170 amino acids in length, or greater than or equal to 160 amino acids in length within the partial RRE region, compared to the unmodified retroviral or lentiviral (e.g., SIV) RNA sequence from which the modified retroviral or lentiviral (e.g., SIV) RNA sequence is derived. Thus, the vector genome plasmid used to generate the modified retroviral or lentiviral (e.g., SIV) vector of the present invention may lack any ORFs (other than a transgene) encoding polypeptides greater than or equal to 160 amino acids in length in the partial RRE region described above, compared to the unmodified plasmid genome vector from which the modified retroviral or lentiviral (e.g., SIV) RNA sequence is derived.

[0153] Alternatively or additionally, one or more retroviral or lentiviral (e.g., SIV) ORFs to be removed may be (at least partially) contained in the RRE sequence. Preferably, one or more retroviral or lentiviral (e.g., SIV) ORFs are (at least partially) contained in the partial RRE sequence. Thus, a retroviral or lentiviral (e.g., SIV) RNA sequence may be modified to reduce the number of ORFs contained (at least partially) in the partial RRE sequence compared to the unmodified retroviral or lentiviral (e.g., SIV) RNA sequence from which the modified retroviral or lentiviral (e.g., SIV) RNA sequence is derived. Thus, the vector genome plasmid used to generate the modified retroviral or lentiviral (e.g., SIV) vector of the present invention may have a reduced number of ORFs contained (at least partially) in the partial RRE sequence compared to the unmodified plasmid genome vector from which the modified retroviral RNA sequence is derived.

[0154] Alternatively or additionally, one or more retroviral or lentiviral (e.g., SIV) ORFs may be contained (at least in part) in the partial Gag sequence. Thus, the retroviral or lentiviral (e.g., SIV) RNA sequence may be modified to reduce the number of ORFs contained (at least in part) in the partial Gag sequence compared to the unmodified retroviral or lentiviral (e.g., SIV) RNA sequence from which the modified retroviral or lentiviral (e.g., SIV) RNA sequence is derived. Thus, the vector genome plasmid used to generate the modified retroviral or lentiviral (e.g., SIV) vector of the invention may have a reduced number of ORFs contained (at least in part) in the partial Gag sequence compared to the unmodified plasmid genome vector from which the modified retroviral RNA sequence is derived.

[0155] Reference herein to an ORF contained in a region of a retroviral / lentiviral (e.g., SIV) sequence, e.g., contained in a partial Gag sequence or a partial RRE sequence, also applies equally and unconditionally to an ORF contained partially in said region of a retroviral / lentiviral (e.g., SIV) sequence, e.g., contained in a partial Gag sequence or a partial RRE sequence, unless expressly stated to the contrary. The ORF to be removed may pass through different regions of the retroviral / lentiviral (e.g., SIV) sequence or may be contained by two or more regions of the retroviral / lentiviral (e.g., SIV) sequence. For example, the ORF to be removed may pass through a partial Gag sequence and enter a partial RRE sequence.

[0156] Typically, removal of one or more retroviral or lentiviral (e.g., SIV) ORFs does not negatively affect expression of a downstream transgene compared to an unmodified retroviral or lentiviral (e.g., SIV) RNA sequence. Removal of one or more retroviral or lentiviral (e.g., SIV) ORFs may increase expression of a downstream transgene compared to an unmodified retroviral or lentiviral (e.g., SIV) RNA sequence. Unmodified retroviral RNA sequences may be generated from unmodified plasmid genome vectors as described above.

[0157] The modified retroviral or lentiviral (e.g., SIV) RNA sequence may be free of ORFs other than the transgene (particularly retroviral or lentiviral (e.g., SIV) ORFs), although this is not required. Rather, the modified retroviral or lentiviral (e.g., SIV) RNA sequence may still include ORFs other than the transgene (including retroviral or lentiviral (e.g., SIV) ORFs), but may include a reduced number of non-transgene ORFs, compared to the unmodified retroviral or lentiviral (e.g., SIV) RNA sequence from which the modified retroviral or lentiviral (e.g., SIV) RNA sequence is derived. Alternatively, or in addition, the length of the remaining non-transgene ORFs may be reduced compared to the unmodified retroviral or lentiviral (e.g., SIV) RNA sequence from which the modified retroviral or lentiviral (e.g., SIV) RNA sequence is derived. Thus, a vector genome plasmid used to generate a modified retroviral or lentiviral (e.g., SIV) vector of the invention may have a reduced number of non-transgene ORFs compared to the unmodified plasmid genome (pDNA1) from which it is derived. Alternatively, or in addition, the remaining non-transgene ORFs in a vector genome plasmid used to generate a modified retroviral or lentiviral (e.g., SIV) vector of the invention may be reduced in length compared to the unmodified retroviral or lentiviral (e.g., SIV) RNA sequence from which the modified retroviral or lentiviral (e.g., SIV) RNA sequence is derived.

[0158] Preferred modifications to reduce the number of ORFs, particularly retroviral / lentiviral (e.g., SIV) ORFs, may comprise or consist of one or more of: (i) insertion of a nucleic acid (e.g., U in the retroviral / lentiviral RNA sequence or T in the corresponding proviral DNA sequence) to destroy the start codon; (ii) substitution of A for U in the retroviral / lentiviral RNA sequence (or A for T in the corresponding proviral DNA sequence) to destroy the start codon; and / or (iii) substitution of U for A in the retroviral / lentiviral RNA sequence (or T for A in the corresponding proviral DNA sequence) to destroy the start codon.

[0159] As exemplified, such preferred modifications to reduce the number of ORFs, particularly retroviral / lentiviral (e.g., SIV) ORFs, include: (i) the introduction of a U in the retroviral / lentiviral RNA sequence immediately 3' to residue 1183 of SEQ ID NO:25 or a T in the corresponding proviral DNA sequence (such an insertion corresponds to residue 1184 of SEQ ID NO:19 and residue 431 of SEQ ID NO:1); (ii) the introduction of a U in the retroviral / lentiviral RNA sequence immediately 3' to residue 1287 of SEQ ID NO:25 or a T in the corresponding proviral DNA sequence (such an insertion corresponds to residue 1289 of SEQ ID NO:19 and residue 536 of SEQ ID NO:1); (iii) the introduction of a U in the retroviral / lentiviral RNA sequence immediately 3' to residue 1303 of SEQ ID NO:25 or a T in the corresponding proviral DNA sequence (such an insertion corresponds to residue 1306 of SEQ ID NO:19 and residue 553 of SEQ ID NO:1); (iv) the introduction of a U in the retroviral / lentiviral RNA sequence immediately 3' to residue 1625 of SEQ ID NO:25 or a T in the corresponding proviral DNA sequence (such an insertion corresponds to residue 1625 of SEQ ID NO:25). (v) substitution of A for U in the retroviral / lentiviral RNA sequence at residue 1787 of SEQ ID NO:25 or substitution of A for T in the corresponding proviral DNA sequence (such insertion corresponds to residue 1794 of SEQ ID NO:19 and residue 1041 of SEQ ID NO:1); (vi) substitution of U for A in the retroviral / lentiviral RNA sequence at residue 2064 of SEQ ID NO:25 or substitution of T for A in the corresponding proviral DNA sequence (corresponding to residue 2071 of SEQ ID NO:19 and residue 1318 of SEQ ID NO:1); and / or (vii) substitution of U for A in the retroviral / lentiviral RNA sequence at residue 2238 of SEQ ID NO:25 or substitution of T for A in the corresponding proviral DNA sequence (corresponding to residue 2245 of SEQ ID NO:19 and residue 1492 of SEQ ID NO:1).

[0160] Particularly preferred modifications for reducing the number of ORFs, in particular retroviral / lentiviral (e.g., SIV) ORFs, are modifications that comprise or consist of a combination of: (i) insertion of a nucleic acid (e.g., U in the retroviral / lentiviral RNA sequence or T in the corresponding proviral DNA sequence) to disrupt one or more start codons (e.g., 2, 3, or 4, preferably 4 start codons); (ii) substitution of A for U in the retroviral / lentiviral RNA sequence (or A for T in the corresponding proviral DNA sequence) to disrupt one or more start codons; and / or (iii) substitution of U for A in the retroviral / lentiviral RNA sequence (or T for A in the corresponding proviral DNA sequence) to disrupt one or more start codons (e.g., 2, 3, or 4, preferably 2 start codons).As exemplified, particularly preferred modifications to remove one or more retroviral / lentiviral (e.g., SIV) ORFs include: (i) the introduction of a U in the retroviral / lentiviral RNA sequence immediately 3' to residue 1183 of SEQ ID NO:25 or a T in the corresponding proviral DNA sequence (such an insertion corresponds to residue 1184 of SEQ ID NO:19 and residue 431 of SEQ ID NO:1); (ii) the introduction of a U in the retroviral / lentiviral RNA sequence immediately 3' to residue 1287 of SEQ ID NO:25 or a T in the corresponding proviral DNA sequence (such an insertion corresponds to residue 1289 of SEQ ID NO:19 and residue 536 of SEQ ID NO:1); (iii) the introduction of a U in the retroviral / lentiviral RNA sequence immediately 3' to residue 1303 of SEQ ID NO:25 or a T in the corresponding proviral DNA sequence (such an insertion corresponds to residue 1306 of SEQ ID NO:19 and residue 553 of SEQ ID NO:1); (iv) the introduction of a U in the retroviral / lentiviral RNA sequence immediately 3' to residue 1625 of SEQ ID NO:25 or a T in the corresponding proviral DNA sequence (such an insertion corresponds to residue 1625 of SEQ ID NO:19 and residue 553 of SEQ ID NO:1); (v) a substitution of A for U in the retroviral / lentiviral RNA sequence or a substitution of A for T in the corresponding proviral DNA sequence at residue 1787 of SEQ ID NO:25 (corresponding to residue 1794 of SEQ ID NO:19 and residue 1041 of SEQ ID NO:1); (vi) a substitution of U for A in the retroviral / lentiviral RNA sequence at residue 2064 of SEQ ID NO:25 or a substitution of T for A in the corresponding proviral DNA sequence (corresponding to residue 2071 of SEQ ID NO:19 and residue 1318 of SEQ ID NO:1); and (vii) a substitution of U for A in the retroviral / lentiviral RNA sequence at residue 2238 of SEQ ID NO:25 or a substitution of T for A in the corresponding proviral DNA sequence (corresponding to residue 2245 of SEQ ID NO:19 and residue 1492 of SEQ ID NO:1).

[0161] As a specific, non-limiting example, modifications to a modified retroviral or lentiviral (e.g., SIV) RNA sequence may remove a retroviral or lentiviral (e.g., SIV) ORF contained (at least in part) within the partial Gag region of the retroviral or lentiviral (e.g., SIV) RNA sequence and / or reduce the size of one or more retroviral or lentiviral (e.g., SIV) ORFs within said region. Preferably, a modified retroviral or lentiviral (e.g., SIV) RNA sequence of the invention is modified so that it does not contain any retroviral or lentiviral (e.g., SIV) ORF encoding a polypeptide longer than 100 amino acids, typically longer than 70 amino acids, within the partial Gag region. Preferably, a modified retroviral or lentiviral (e.g., SIV) RNA sequence of the invention is modified so that it does not contain any retroviral or lentiviral (e.g., SIV) ORF encoding a polypeptide longer than 200 amino acids, typically longer than 160 amino acids, within the partial RRE region. Particularly preferred are modified retroviral or lentiviral (e.g., SIV) RNA sequences of the present invention that have been modified so as not to contain (i) any retroviral or lentiviral (e.g., SIV) ORFs encoding polypeptides longer than 100 amino acids, typically longer than 70 amino acids, within the partial Gag region; and (ii) any retroviral or lentiviral (e.g., SIV) ORFs encoding polypeptides longer than 200 amino acids, typically longer than 160 amino acids, within the partial RRE region. The present invention provides retroviral or lentiviral (e.g., SIV) vectors comprising the modified retroviral or lentiviral (e.g., SIV) RNA sequences.

[0162] Any modification or combination thereof to reduce the number of ORFs, particularly retroviral or lentiviral (e.g., SIV) ORFs, within a retroviral or lentiviral (e.g., SIV) RNA sequence of the present invention may be used in combination with any codon substitution modification or combination thereof described herein.

[0163] Thus, the present invention provides modified retroviral or lentiviral (e.g., SIV) RNA sequences that (a) do not contain (i) any retroviral or lentiviral (e.g., SIV) ORFs encoding polypeptides longer than 100 amino acids, typically longer than 70 amino acids, within the partial Gag region; (ii) any retroviral or lentiviral (e.g., SIV) ORFs encoding polypeptides longer than 200 amino acids, typically longer than 160 amino acids, within the partial RRE region; and (b) the codon substitutions comprise or consist of a combination of (i) a frameshift mutation and replacement of a stop codon in the Env ORF of the retroviral / lentiviral RNA sequence; and (ii) the introduction of an Sbfl restriction site, which may optionally replace the EcoRl restriction site within the retroviral / lentiviral RNA sequence, particularly the specific examples described herein. The present invention also provides retroviral or lentiviral (e.g., SIV) vectors comprising the modified retroviral or lentiviral (e.g., SIV) RNA sequence.

[0164] Any codon substitution or combination thereof may be used in combination with any modification or combination thereof to reduce the number of ORFs, particularly retroviral / lentiviral (e.g., SIV) ORFs, where (a) the codon substitution comprises or consists of a combination of (i) a frameshift mutation and replacement of a stop codon into the Env ORF of the retroviral / lentiviral RNA sequence; and (ii) the introduction of an Sbfl restriction site, which may optionally replace an EcoRl restriction site within the retroviral / lentiviral RNA sequence; and (b) the modification to reduce the number of ORFs, particularly retroviral / lentiviral (e.g., SIV) ORFs, comprises (i) a nucleic acid (e.g., U in the retroviral / lentiviral RNA sequence or a nucleotide sequence in the corresponding proviral DNA sequence) to destroy one or more start codons (e.g., 2, 3, or 4, preferably 4 start codons). Preferred are retroviral / lentiviral (e.g., SIV) RNA sequences that comprise or consist of a combination of: (ii) an insertion of a U in the retroviral / lentiviral RNA sequence (or a T in the corresponding proviral DNA sequence) to destroy one or more start codons; and (iii) a substitution of a U in the retroviral / lentiviral RNA sequence (or a T in the corresponding proviral DNA sequence) to destroy one or more start codons (e.g., 2, 3, or 4, preferably 2 start codons).

[0165] (a) the codon substitutions comprise or consist of a combination of: (i) replacement of the motif corresponding to residues 2347-2352 of SEQ ID NO:25 with the motif corresponding to residues 2354-2360 of SEQ ID NO:25; and (ii) replacement of the motif corresponding to residues 1734-1739 of SEQ ID NO:25 with the motif corresponding to residues 1738-1746 of SEQ ID NO:25; and (b) the modifications to reduce the number of ORFs, particularly retroviral / lentiviral (e.g., SIV) ORFs, comprise: (i) replacement of residues 1183-1189 of SEQ ID NO:25 with the motif corresponding to residues 1183-1189 of SEQ ID NO:25. (ii) the introduction of a U in the retroviral / lentiviral RNA sequence immediately 3' to residue 1184 of SEQ ID NO:19 or a T in the corresponding proviral DNA sequence (such an insertion corresponds to residue 1184 of SEQ ID NO:19 and residue 431 of SEQ ID NO:1); (ii) the introduction of a U in the retroviral / lentiviral RNA sequence immediately 3' to residue 1287 of SEQ ID NO:25 or a T in the corresponding proviral DNA sequence (such an insertion corresponds to residue 1289 of SEQ ID NO:19 and residue 536 of SEQ ID NO:1); (iii) the sequence (iv) introduction of a U in the retroviral / lentiviral RNA sequence immediately 3' to residue 1303 of SEQ ID NO:25 or a T in the corresponding proviral DNA sequence (such an insertion corresponds to residue 1306 of SEQ ID NO:19 and residue 553 of SEQ ID NO:1); (iv) introduction of a U in the retroviral / lentiviral RNA sequence immediately 3' to residue 1625 of SEQ ID NO:25 or a T in the corresponding proviral DNA sequence (such an insertion corresponds to residue 1629 of SEQ ID NO:19 and residue 876 of SEQ ID NO:1); (v) substitution of an A for an A in the retroviral / lentiviral RNA sequence at residue 1787 of SEQ ID NO:25 or a T for an A in the corresponding proviral DNA sequence (corresponding to residue 1794 of SEQ ID NO:19 and residue 1041 of SEQ ID NO:1); (vi) substitution of an A for a U in the retroviral / lentiviral RNA sequence at residue 2064 of SEQ ID NO:25 or a T for an A in the corresponding proviral DNA sequence (corresponding to residue 2071 of SEQ ID NO:19 and residue 1318 of SEQ ID NO:1);and (vii) a combination of a U for A substitution in the retroviral / lentiviral RNA sequence at residue 2238 of SEQ ID NO:25 or a T for A substitution in the corresponding proviral DNA sequence (corresponding to residue 2245 of SEQ ID NO:19 and residue 1492 of SEQ ID NO:1) is particularly preferred;

[0166] Particularly preferably, the present invention also provides an SIV vector pseudotyped with Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) proteins, wherein (a) the vector comprises a modified retroviral RNA sequence comprising or consisting of the nucleic acid sequence of SEQ ID NO: 1, preferably the modified retroviral RNA sequence consisting of the nucleic acid sequence of SEQ ID NO: 1; and (b) the F protein comprises a first subunit comprising or consisting of the amino acid sequence of SEQ ID NO: 14, and a second subunit comprising or consisting of the amino acid sequence of SEQ ID NO: 15. The vector may further comprise one or more of: (a) a p17 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 2; (b) a p24 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 3; (c) a p8 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 4; (d) a protease comprising or consisting of the amino acid sequence of SEQ ID NO: 5; (e) a p51 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 6; (f) a p15 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 7; (g) a p31 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 8; (h) a Gag protein comprising or consisting of the amino acid sequence of SEQ ID NO: 9; and / or (i) a Pol protein comprising or consisting of the amino acid sequence of SEQ ID NO: 10. Optionally, the vector may comprise each of (a) to (g) and further comprise one or both of (h) and (i).

[0167] The retroviral / lentiviral (e.g., SIV) RNA sequences of the present invention may contain one or more additional modifications in addition to the codon substitutions and / or modifications to reduce the retroviral / lentiviral (e.g., SIV) ORF described herein. As a non-limiting example, the retroviral / lentiviral (e.g., SIV) RNA sequence may be CpG-depleted (or CpG-free) to promote gene expression. Standard techniques for modifying transgene sequences in this manner are known in the art.

[0168] As exemplified herein, retroviral / lentiviral (e.g., SIV) vectors comprising the modified retroviral / lentiviral (e.g., SIV) RNA sequences of the present invention at least maintained and potentially increased transgene expression; and / or at least maintained and potentially increased integration of the retroviral / lentiviral (e.g., SIV) RNA sequence into target cells. Retroviral / lentiviral (e.g., SIV) vectors comprising the modified retroviral / lentiviral (e.g., SIV) RNA sequences of the present invention also typically at least maintained and potentially increased vector yield compared to retroviral / lentiviral (e.g., SIV) vectors comprising the unmodified retroviral / lentiviral (e.g., SIV) RNA sequence from which the modified retroviral / lentiviral (e.g., SIV) RNA sequence is derived. This effect on vector yield can be further increased by the use of codon-optimized GagPol, as described herein.

[0169] Retroviral / lentiviral (e.g., SIV) vectors contain a promoter operably linked to a transgene, enabling expression of the transgene. Typically, the promoter is a hybrid human CMV enhancer / EF1a (hCEF) promoter. This hCEF promoter may lack the intron corresponding to nucleotides 570-709 and the exon corresponding to nucleotides 728-733 of the hCEF promoter. A preferred example of an hCEF promoter sequence of the present invention is provided by SEQ ID NO: 26. The promoter may also be a CMV promoter. An example of a CMV promoter sequence is provided by SEQ ID NO: 27. The promoter may also be a human elongation factor 1a (EF1a) promoter. An example of an EF1a promoter is provided by SEQ ID NO: 28. Other promoters for transgene expression are known in the art, and their suitability for the retroviral / lentiviral (e.g., SIV) vectors of the present invention is determined using routine techniques known in the art. Non-limiting examples of other promoters include UbC and UCOE. As described herein, the promoter may be modified to further regulate expression of the transgene of the present invention.

[0170] The promoter contained in the retroviral / lentiviral (e.g., SIV) vector of the present invention may be specifically selected and / or modified to further refine the regulation of therapeutic gene expression. Suitable promoters and standard techniques for such modifications are known in the art. As a non-limiting example, some (CpG-free) promoters suitable for use in the present invention are described in Pringle et al. (J. Mol. Med. Berl. 2012, 90(12): 1487-96), which is incorporated herein by reference in its entirety. Preferably, the retroviral / lentiviral vector of the present invention (particularly the SIV F / HN vector) comprises an hCEF promoter with a low CpG dinucleotide content or no CpG dinucleotides. The hCEF promoter may have all CG dinucleotides replaced with any one of AG, TG, or GT. Therefore, the hCEF promoter may be CpG-free. A preferred example of a CpG-free hCEF promoter sequence of the present invention is provided by SEQ ID NO: 26. The absence of CpG dinucleotides typically further improves the performance of the retroviral / lentiviral (e.g., SIV) vectors of the invention, particularly in situations where it is undesirable to induce an immune response to the expressed antigen or an inflammatory response to the delivered expression construct. The elimination of CpG dinucleotides reduces the appearance of flu-like symptoms and inflammation that can result from administration of the construct, particularly when administered to the respiratory tract.

[0171] Retroviral / lentiviral (for example, SIV) vector of the present invention can be modified to allow gene expression to be shut down.The standard technique for modifying vector in this way is known in the art.As a non-limiting example, Tet-responsive promoter is widely used.

[0172] The retroviral / lentiviral (e.g., SIV) vectors of the present invention may contain a transgene encoding a therapeutic polypeptide or protein for the treatment of such diseases, particularly diseases or disorders of the airway, respiratory tract, or lungs.

[0173] Thus, the retroviral / lentiviral (e.g., SIV) vectors of the invention may comprise a transgene encoding (i) a secreted therapeutic protein, optionally alpha-1 antitrypsin (A1AT), factor VIII, surfactant protein B (SFTPB), factor VII, factor IX, factor X, factor XI, von Willebrand factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), and a monoclonal antibody against an infectious pathogen; or (ii) a protein selected from CFTR, ABCA3, DNAH5, DNAH11, DNAI1, and DNAI2. Other examples of transgenes that can be included in the retroviral / lentiviral (e.g., SIV) vectors of the invention include genes related to or associated with other surfactant deficiencies.

[0174] The transgene contained in the vector of the present invention may be modified to enhance expression. For example, the transgene sequence may be CpG-depleted (or CpG-free) and / or further modified to enhance gene expression. Standard techniques for modifying transgene sequences in this manner are known in the art.

[0175] Preferably, the transgene encodes CFTR. An example of a CFTR cDNA is provided by SEQ ID NO: 29. Variants thereof (as described herein) also include, inter alia, variants having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100%, identity to SEQ ID NO: 29. Preferably, the CFTR transgene has at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 29.

[0176] The transgene may encode A1AT. An example of an A1AT transgene is provided by SEQ ID NO: 30, or the complementary sequence of SEQ ID NO: 31. SEQ ID NO: 30 is a codon-optimized, CpG-depleted A1AT transgene previously designed by the present inventors to enhance translation in human cells. Such optimization has been shown to enhance gene expression by up to 15-fold. Variants of the same sequence (as defined herein) with the same technical effect of enhancing translation compared to the unmodified (wild-type) A1AT gene sequence are also encompassed by the present invention. The polypeptide encoded by the A1AT transgene may be exemplified by the polypeptide of SEQ ID NO: 32. Variants thereof (as described herein) also include, inter alia, variants having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or more, up to 100% identity to SEQ ID NO: 30, 31 or 32. Preferably, the A1AT variant has at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 30, 31 or 32.

[0177] The transgene may encode FVIII. Examples of FVIII transgenes are provided by SEQ ID NOs: 33 and 34, or by the respective complementary sequences of SEQ ID NOs: 35 and 36. The polypeptide encoded by the FVIII transgene may be exemplified by the polypeptide of SEQ ID NO: 37 or 38. Variants thereof (as described herein) also include, in particular, variants having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100%, identity to any one of SEQ ID NOs: 33-38. Preferably, the FVIII variant has at least 90%, at least 95%, or at least 99% identity to any one of SEQ ID NOs: 33-38.

[0178] The transgene of the present invention may be any one or more of DNAH5, DNAH11, DNAI1, and DNAI2, or other known related genes.

[0179] When respiratory airway epithelia are targeted for delivery of retroviral / lentiviral (e.g., SIV) vectors, the transgene may encode A1AT, SFTPB, or GM-CSF. The transgene may encode a monoclonal antibody (mAb) against an infectious pathogen. The transgene may encode an anti-TNF alpha. The transgene may encode a therapeutic protein involved in inflammatory, immune, or metabolic conditions.

[0180] The retroviral / lentiviral (e.g., SIV) vectors of the invention may be delivered to cells of the respiratory tract to allow for the production of proteins that are secreted into the circulatory system. In such embodiments, the transgene may encode Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, and / or von Willebrand factor. Such vectors may be used in the treatment of diseases, particularly cardiovascular diseases and blood disorders, preferably blood clotting deficiencies such as hemophilia. The transgene may also encode a mAb against an infectious pathogen or a protein involved in an inflammatory, immune, or metabolic condition, e.g., a lysosomal storage disease.

[0181] The retroviral / lentiviral (e.g., SIV) vectors of the present invention may not have an intron located between the promoter and the transgene. Similarly, in the vector genome (pDNA1) plasmid (e.g., pGM830 described herein, having the sequence of SEQ ID NO: 20), there may be no intron between the promoter and the transgene.

[0182] In some preferred embodiments, a retroviral / lentiviral (e.g., SIV) vector comprises a hCEF promoter and a CFTR transgene, including those described herein. Optionally, the retroviral / lentiviral (e.g., SIV) vector may lack an intron located between the promoter and the transgene. Such retroviral / lentiviral (e.g., SIV) vectors may be generated by the methods described herein using a genomic plasmid carrying the CFTR transgene and promoter.

[0183] In some preferred embodiments, a retroviral / lentiviral (e.g., SIV) vector comprises an hCEF promoter and an A1AT transgene, including those described herein. Optionally, the retroviral / lentiviral (e.g., SIV) vector may lack an intron located between the promoter and the transgene. Such retroviral / lentiviral (e.g., SIV) vectors may be generated by the methods described herein using a genomic plasmid carrying the A1AT transgene and promoter.

[0184] In some preferred embodiments, the retroviral / lentiviral (e.g., SIV) vector comprises an hCEF or CMW promoter and a FVIII transgene, including those described herein. Optionally, the retroviral / lentiviral (e.g., SIV) vector may lack an intron located between the promoter and the transgene. Such retroviral / lentiviral (e.g., SIV) vectors may be generated by the methods described herein using a genomic plasmid carrying the FVIII transgene and promoter.

[0185] The retroviral / lentiviral (e.g., SIV) vectors described herein comprise a transgene, which comprises a nucleic acid sequence that encodes a gene product, e.g., a protein, particularly a therapeutic protein.

[0186] For example, in one embodiment, the nucleic acid sequence encoding CFTR, A1AT, or FVIII comprises (or consists of) a nucleic acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100%, sequence identity to a CFTR, A1AT, or FVIII nucleic acid sequence, respectively, examples of which are described herein. In a further embodiment, the nucleic acid sequence encoding CFTR, A1AT, or FVIII comprises (or consists of) a nucleic acid sequence having at least 95% (such as at least 95, 96, 97, 98, 99, or 100%) sequence identity to a CFTR, A1AT, or FVIII nucleic acid sequence, respectively, examples of which are described herein. In one embodiment, the nucleic acid sequence encoding CFTR is provided by SEQ ID NO: 29, the nucleic acid sequence encoding A1AT is provided by SEQ ID NO: 30 or by the complementary sequence of SEQ ID NO: 31, and / or the nucleic acid sequence encoding FVIII is provided by SEQ ID NOs: 33 and 34, or by the complementary sequences of SEQ ID NOs: 35 and 36, respectively, or variants thereof.

[0187] The amino acid sequence of the CFTR, A1AT or FVIII transgene may comprise (or consist of) an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or more, up to 100%, preferably at least 90%, at least 95%, or at least 99% sequence identity to a functional CFTR, A1AT or FVIII polypeptide sequence, respectively.

[0188] Retroviral / lentiviral (e.g., SIV) vectors of the invention may include a central polypurine tract (cPPT) and / or a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). An exemplary WPRE sequence is provided by SEQ ID NO:39.

[0189] As described herein, retroviral / lentiviral (e.g., SIV) RNA sequences are derived from proviral DNA sequences. The proviral DNA sequences are themselves provided during the manufacturing process by the vector genome plasmid pDNA1. However, retroviral / lentiviral (e.g., SIV) RNA sequences are not identical to the proviral DNA sequences (and therefore are not identical to the vector genome plasmid pDNA1). Rather, retroviral / lentiviral (e.g., SIV) RNA sequences are shorter in length than the corresponding proviral DNA sequences, and the exact limits or boundaries of retroviral / lentiviral (e.g., SIV) RNA sequences are typically not easily determined. In other words, it is generally not possible to identify the exact retroviral / lentiviral (e.g., SIV) RNA sequence (with specifically specified 5' and 3' ends) simply from the primary sequence of the proviral DNA sequence (and therefore the sequence of the vector genome plasmid pDNA1).

[0190] Retroviral / lentiviral (e.g., SIV) vectors typically comprise modified retroviral / lentiviral (e.g., SIV) RNA sequences that are less than 10,000 bases in length, less than 9,000 bases in length, or less than 8,000 bases in length. Preferably, retroviral / lentiviral (e.g., SIV) vectors comprise modified retroviral / lentiviral (e.g., SIV) RNA sequences that are less than 9,000 bases in length.

[0191] A retroviral / lentiviral (e.g., SIV) vector may comprise a modified retroviral / lentiviral (e.g., SIV) RNA sequence that comprises, or consists of, a nucleic acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100%, identity to SEQ ID NO: 1. The modified retroviral / lentiviral (e.g., SIV) RNA sequence may comprise, or consist of, a nucleic acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 1. The modified retroviral / lentiviral (e.g., SIV) RNA sequence may comprise, or consist of, a nucleic acid sequence having at least 99% identity to SEQ ID NO: 1. The modified retroviral sequence may comprise, or consist of, the nucleic acid sequence of SEQ ID NO: 1.

[0192] The present invention provides retroviral / lentiviral (e.g., SIV) vectors comprising a retroviral / lentiviral (e.g., SIV) RNA sequence consisting of a nucleic acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100%, identity to SEQ ID NO: 1. The modified retroviral / lentiviral (e.g., SIV) RNA sequence may consist of a nucleic acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 1. The modified retroviral / lentiviral (e.g., SIV) RNA sequence may consist of a nucleic acid sequence having at least 99% identity to SEQ ID NO: 1. The present invention provides retroviral / lentiviral (e.g., SIV) vectors comprising a retroviral / lentiviral (e.g., SIV) RNA sequence consisting of the nucleic acid sequence of SEQ ID NO: 1.

[0193] The retroviral / lentiviral (e.g., SIV) vector may comprise a modified retroviral / lentiviral (e.g., SIV) RNA sequence that (a) is less than 10,000 bases in length, less than 9,000 bases in length, or less than 8,000 bases in length; and (b) comprises or consists of a nucleic acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100%, identity to SEQ ID NO:1.

[0194] The retroviral / lentiviral (e.g., SIV) vector may comprise a modified retroviral / lentiviral (e.g., SIV) RNA sequence that (a) is less than 10,000 bases in length, less than 9,000 bases in length, or less than 8,000 bases in length; and (b) comprises or consists of a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100%, identity to SEQ ID NO:1.

[0195] The retroviral / lentiviral (e.g., SIV) vector may comprise a modified retroviral / lentiviral (e.g., SIV) RNA sequence that (a) is less than 10,000 bases in length, less than 9,000 bases in length, or less than 8,000 bases in length; and (b) comprises or consists of a nucleic acid sequence having at least 99%, at least 99.5%, at least 99.9%, or more, up to 100%, identity to SEQ ID NO:1.

[0196] The retroviral / lentiviral (e.g., SIV) vector may comprise a modified retroviral / lentiviral (e.g., SIV) RNA sequence that is (a) less than 10,000 bases in length, less than 9,000 bases in length, or less than 8,000 bases in length; and (b) consists of a nucleic acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or more, up to 100%, identity to SEQ ID NO:1.

[0197] The retroviral / lentiviral (e.g., SIV) vector may comprise a modified retroviral / lentiviral (e.g., SIV) RNA sequence that is (a) less than 10,000 bases in length, less than 9,000 bases in length, or less than 8,000 bases in length; and (b) consists of a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100%, identity to SEQ ID NO:1.

[0198] The retroviral / lentiviral (e.g., SIV) vector may comprise a modified retroviral / lentiviral (e.g., SIV) RNA sequence that is (a) less than 10,000 bases in length, less than 9,000 bases in length, or less than 8,000 bases in length; and (b) consists of a nucleic acid sequence having at least 99%, at least 99.5%, at least 99.9%, or more, up to 100%, identity to SEQ ID NO:1.

[0199] The retroviral / lentiviral (e.g., SIV) vector may comprise a modified retroviral / lentiviral (e.g., SIV) RNA sequence that (a) is less than 9,000 bases in length, or less than 8,000 bases in length; and (b) comprises or consists of a nucleic acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100%, identity to SEQ ID NO:1.

[0200] The retroviral / lentiviral (e.g., SIV) vector may comprise a modified retroviral / lentiviral (e.g., SIV) RNA sequence that (a) is less than 9,000 bases in length, or less than 8,000 bases in length; and (b) comprises or consists of a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100%, identity to SEQ ID NO:1.

[0201] The retroviral / lentiviral (e.g., SIV) vector may comprise a modified retroviral / lentiviral (e.g., SIV) RNA sequence that (a) is less than 9,000 bases in length, or less than 8,000 bases in length; and (b) comprises or consists of a nucleic acid sequence having at least 99%, at least 99.5%, at least 99.9%, or more, up to 100%, identity to SEQ ID NO:1.

[0202] The retroviral / lentiviral (e.g., SIV) vector may comprise a modified retroviral / lentiviral (e.g., SIV) RNA sequence that is (a) less than 9,000 bases in length, or less than 8,000 bases in length; and (b) consists of a nucleic acid sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or more, up to 100%, identity to SEQ ID NO:1.

[0203] The retroviral / lentiviral (e.g., SIV) vector may comprise a modified retroviral / lentiviral (e.g., SIV) RNA sequence that is (a) less than 9,000 bases in length, or less than 8,000 bases in length; and (b) consists of a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or more, up to 100%, identity to SEQ ID NO:1.

[0204] The retroviral / lentiviral (e.g., SIV) vector may comprise a modified retroviral / lentiviral (e.g., SIV) RNA sequence that is (a) less than 9,000 bases in length, or less than 8,000 bases in length; and (b) consists of a nucleic acid sequence that has at least 99%, at least 99.5%, at least 99.9%, or more, up to 100%, identity to SEQ ID NO:1.

[0205] Preferably, the retroviral / lentiviral (e.g., SIV) vector comprises a modified retroviral / lentiviral (e.g., SIV) RNA sequence that (a) is less than 9,000 bases in length; and (b) comprises, or consists of, a nucleic acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% identity to SEQ ID NO: 1. More preferably, the retroviral / lentiviral (e.g., SIV) vector comprises a modified retroviral / lentiviral (e.g., SIV) RNA sequence that (a) is less than 9,000 bases in length; and (b) comprises, or consists of, a nucleic acid sequence having at least 99% identity to SEQ ID NO: 1. Even more preferably, the retroviral / lentiviral (e.g., SIV) vector comprises a modified retroviral / lentiviral (e.g., SIV) RNA sequence that (a) is less than 9,000 bases in length; and (b) consists of a nucleic acid sequence having at least 99% identity to SEQ ID NO: 1. Even more preferably, the retroviral / lentiviral (e.g., SIV) vector comprises a modified retroviral / lentiviral (e.g., SIV) RNA sequence that (a) is less than 9,000 bases in length; and (b) comprises or consists of the nucleic acid sequence of SEQ ID NO: 1. Even more preferably, the retroviral / lentiviral (e.g., SIV) vector comprises a modified retroviral / lentiviral (e.g., SIV) RNA sequence that (a) is less than 9,000 bases in length; and (b) consists of the nucleic acid sequence of SEQ ID NO: 1.

[0206] The 5' and / or 3' limits of the modified retroviral / lentiviral (e.g., SIV) RNA sequence may each independently allow for some degree of flexibility, such that the 5' end of the modified retroviral / lentiviral (e.g., SIV) RNA sequence may not correspond to the first nucleotide of SEQ ID NO:1, and / or the 3' end of the modified retroviral / lentiviral (e.g., SIV) RNA sequence may not correspond to the last nucleotide of SEQ ID NO:1.

[0207] Thus, a modified retroviral / lentiviral (e.g., SIV) RNA sequence may comprise up to an additional 200 nucleotides, up to an additional 150 nucleotides, up to an additional 100 nucleotides, up to an additional 75 nucleotides, up to an additional 50 nucleotides, up to an additional 25 nucleotides, up to an additional 10 nucleotides, up to an additional 5 nucleotides at the 5' and / or 3' end, for example, compared to SEQ ID NO: 1. A modified retroviral / lentiviral (e.g., SIV) RNA sequence may comprise an additional 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotide at the 5' and / or 3' end, for example, compared to SEQ ID NO: 1. The presence and number of additional nucleotides at the 5' end of a modified retroviral / lentiviral (e.g., SIV) RNA sequence is independent of the presence and number of additional nucleotides at the 3' end of the modified retroviral / lentiviral (e.g., SIV) RNA sequence. As a non-limiting example, a modified retroviral / lentiviral (e.g., SIV) RNA sequence may include up to 3 additional nucleotides at the 5' end and up to 200 additional nucleotides at the 3' end, e.g., compared to SEQ ID NO: 1. As a further non-limiting example, a modified retroviral / lentiviral (e.g., SIV) RNA sequence may include no additional nucleotides at the 5' end or may include an additional 42 nucleotides at the 3' end, e.g., compared to SEQ ID NO: 1. Preferably, a modified retroviral / lentiviral (e.g., SIV) RNA sequence does not include any additional nucleotides at the 5' end, but may include up to 200 additional nucleotides at the 3' end (as described above), e.g., compared to SEQ ID NO: 1.

[0208] The modified retroviral / lentiviral (e.g., SIV) RNA sequence may contain at most 200 fewer nucleotides, at most 150 fewer nucleotides, at most 100 fewer nucleotides, at most 75 fewer nucleotides, at most 50 fewer nucleotides, at most 25 fewer nucleotides, at most 10 fewer nucleotides, at most 5 fewer nucleotides at the 5' and / or 3' end, for example compared to SEQ ID NO: 1. The modified retroviral / lentiviral (e.g., SIV) RNA sequence may contain at most 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 fewer nucleotides at the 5' and / or 3' end, for example compared to SEQ ID NO: 1. The number of deleted nucleotides at the 5' end of a modified retroviral / lentiviral (e.g., SIV) RNA sequence is independent of the presence and number of deleted nucleotides at the 3' end of the modified retroviral / lentiviral (e.g., SIV) RNA sequence. As a non-limiting example, a modified retroviral / lentiviral (e.g., SIV) RNA sequence may contain up to 3 nucleotides less at the 5' end, e.g., compared to SEQ ID NO: 1, and up to 200 nucleotides at the 3' end, e.g., compared to SEQ ID NO: 1. As a further non-limiting example, a modified retroviral / lentiviral (e.g., SIV) RNA sequence may contain no nucleotides at the 5' end, e.g., compared to SEQ ID NO: 1, or may contain up to 42 nucleotides at the 3' end, e.g., compared to SEQ ID NO: 1. Preferably, a modified retroviral / lentiviral (e.g., SIV) RNA sequence does not contain any nucleotides at the 5' end, e.g., compared to SEQ ID NO: 1, but may contain up to 200 nucleotides less at the 3' end (as described above).

[0209] One end of the modified retroviral / lentiviral (e.g., SIV) RNA sequence may have additional nucleotides, e.g., compared to SEQ ID NO: 1, and the other end may have fewer nucleotides, e.g., compared to SEQ ID NO: 1. Thus, the 5' end may have additional nucleotides, e.g., compared to SEQ ID NO: 1, and the 3' end may have fewer nucleotides, e.g., compared to SEQ ID NO: 1. The 3' end may have additional nucleotides, e.g., compared to SEQ ID NO: 1, and the 5' end may have fewer nucleotides, e.g., compared to SEQ ID NO: 1. The disclosure herein regarding the number of additional and / or deleted nucleotides applies equally and unconditionally to modified retroviral / lentiviral (e.g., SIV) RNA sequences having additional nucleotides at one end, e.g., compared to SEQ ID NO: 1, and fewer nucleotides at the other end, e.g., compared to SEQ ID NO: 1. Preferably, the modified retroviral / lentiviral (e.g., SIV) RNA sequence does not include any additional / missing nucleotides at the 5' end, e.g., compared to SEQ ID NO: 1, but may include additional or fewer nucleotides at the 3' end (as described above).

[0210] As described herein, retroviral / lentiviral (e.g., SIV) vectors having modified retroviral / lentiviral (e.g., SIV) RNA sequences according to the present invention avoid the potential safety risks described herein while (i) maintaining or even increasing transgene expression; (ii) maintaining or even increasing integration of the retroviral / lentiviral (e.g., SIV) RNA sequence into the host cell genome; and / or (iii) maintaining or even increasing retroviral / lentiviral (e.g., SIV) vector yield.

[0211] As such, retroviral / lentiviral (e.g., SIV) vectors comprising the modified retroviral / lentiviral (e.g., SIV) RNA sequences of the present invention typically exhibit high levels of transgene expression. Typically, retroviral / lentiviral (e.g., SIV) vectors having the modified retroviral / lentiviral (e.g., SIV) RNA sequences of the present invention are at least equivalent in terms of transgene expression to retroviral / lentiviral (e.g., SIV) vectors comprising the unmodified retroviral / lentiviral (e.g., SIV) RNA sequence from which the modified retroviral / lentiviral (e.g., SIV) RNA sequence is derived (i.e., the corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence).

[0212] As used herein, the term "equivalent transgene expression" can be defined as a modified retroviral / lentiviral (e.g., SIV) RNA sequence that does not significantly reduce transgene expression of a retroviral / lentiviral (e.g., SIV) vector compared to a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence. As a non-limiting example, transgene expression by a retroviral / lentiviral (e.g., SIV) vector comprising a modified retroviral / lentiviral (e.g., SIV) RNA sequence into a host / target cell genome may be no more than 2-fold lower, no more than 1.5-fold lower, no more than 1.0-fold lower, no more than 0.5-fold lower, no more than 0.25-fold lower, or less than that by a retroviral / lentiviral (e.g., SIV) vector comprising a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence. The term "equivalent transgene expression" may be defined as transgene expression by a retroviral / lentiviral (e.g., SIV) vector comprising a modified retroviral / lentiviral (e.g., SIV) RNA sequence into the host / target cell genome is statistically unchanged (e.g., p<0.05, p<0.01) compared to transgene expression by a retroviral / lentiviral (e.g., SIV) vector comprising a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence.

[0213] Preferably, transgene expression by a retroviral / lentiviral (e.g., SIV) vector comprising a modified retroviral / lentiviral (e.g., SIV) RNA sequence vector into a host / target cell genome is increased compared to transgene expression by a retroviral / lentiviral (e.g., SIV) vector comprising a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence. Transgene expression by a retroviral / lentiviral (e.g., SIV) vector comprising a modified retroviral / lentiviral (e.g., SIV) RNA sequence into a host / target cell genome may be at least 1.5-fold, at least 2-fold, or at least 2.5-fold higher than transgene expression by a retroviral / lentiviral (e.g., SIV) vector comprising a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence.

[0214] Alternatively or additionally, retroviral / lentiviral (e.g., SIV) vectors comprising the modified retroviral / lentiviral (e.g., SIV) RNA sequences of the invention exhibit high levels of vector integration into the host / target cell genome. Typically, retroviral / lentiviral (e.g., SIV) vectors having the modified retroviral / lentiviral (e.g., SIV) RNA sequences of the invention are at least equivalent in terms of integration into the host / target cell genome compared to retroviral / lentiviral (e.g., SIV) vectors comprising the corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequences.

[0215] As used herein, the term "equivalent integration" can be defined as a modified retroviral / lentiviral (e.g., SIV) RNA sequence that does not significantly reduce integration of the retroviral / lentiviral (e.g., SIV) vector into the host / target cell genome compared to the corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence. As a non-limiting example, integration of a retroviral / lentiviral (e.g., SIV) vector comprising a modified retroviral / lentiviral (e.g., SIV) RNA sequence of the present invention into the host / target cell genome may be 2-fold or less lower, 1.5-fold or less lower, 1.0-fold or less lower, 0.5-fold or less lower, 0.25-fold or less lower, or less than the integration of a retroviral / lentiviral (e.g., SIV) vector comprising the corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence into the host / target cell genome. The term "equivalent integration" may be defined such that integration of a retroviral / lentiviral (e.g., SIV) vector comprising a modified retroviral / lentiviral (e.g., SIV) RNA sequence of the present invention into the host / target cell genome is statistically unchanged (e.g., p<0.05, p<0.01) compared to integration of a retroviral / lentiviral (e.g., SIV) vector comprising a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence.

[0216] Preferably, integration of a retroviral / lentiviral (e.g., SIV) vector comprising a modified retroviral / lentiviral (e.g., SIV) RNA sequence vector of the present invention into a host / target cell genome is increased compared to integration of a retroviral / lentiviral (e.g., SIV) vector comprising a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence. Integration of a retroviral / lentiviral (e.g., SIV) vector comprising a modified retroviral / lentiviral (e.g., SIV) RNA sequence of the present invention into a host / target cell genome may be at least 1.5-fold, at least 2-fold, or at least 2.5-fold higher than integration of a retroviral / lentiviral (e.g., SIV) vector comprising a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence.

[0217] Alternatively or additionally, the present invention provides high-titer purified retroviral / lentiviral (e.g., SIV) vectors comprising modified retroviral / lentiviral (e.g., SIV) RNA sequences. Typically, the titer of a retroviral / lentiviral (e.g., SIV) vector having a modified retroviral / lentiviral (e.g., SIV) RNA sequence of the present invention is at least equivalent to the titer of a retroviral / lentiviral (e.g., SIV) vector comprising a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence.

[0218] As used herein, the term "equivalent potency" can be defined as a modified retroviral / lentiviral (e.g., SIV) RNA sequence that does not significantly reduce the titer of a retroviral / lentiviral (e.g., SIV) vector compared to a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence. As a non-limiting example, the titer of a retroviral / lentiviral (e.g., SIV) vector comprising a modified retroviral / lentiviral (e.g., SIV) RNA sequence of the present invention may be no more than 2-fold lower, no more than 1.5-fold lower, no more than 1.0-fold lower, no more than 0.5-fold lower, no more than 0.25-fold lower, or less than the titer of a retroviral / lentiviral (e.g., SIV) vector comprising a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence. The term "equivalent potency" may be defined such that the potency of a retroviral / lentiviral (e.g., SIV) vector comprising a modified retroviral / lentiviral (e.g., SIV) RNA sequence of the present invention is not statistically altered (e.g., p<0.05, p<0.01) compared to the potency of a retroviral / lentiviral (e.g., SIV) vector comprising a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence.

[0219] Preferably, the titer of a retroviral / lentiviral (e.g., SIV) vector comprising a modified retroviral / lentiviral (e.g., SIV) RNA sequence vector of the present invention is increased compared to the titer of a retroviral / lentiviral (e.g., SIV) vector comprising a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence. The titer of a retroviral / lentiviral (e.g., SIV) vector comprising a modified retroviral / lentiviral (e.g., SIV) RNA sequence of the present invention may be at least 1.5-fold, at least 2-fold, or at least 2.5-fold higher than the titer of a retroviral / lentiviral (e.g., SIV) vector comprising a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence.

[0220]

[0013] Producing high titers of retroviral / lentiviral (e.g., SIV) vectors may impart other desirable properties to the resulting vector product. For example, without being bound by theory, production at high titers without the need for significant concentration by methods such as TFF is believed to result in a higher quality vector product than a corresponding retroviral / lentiviral (e.g., SIV) vector bearing an unmodified retroviral / lentiviral (e.g., SIV) RNA sequence, because the vector is exposed to less shear forces that can damage viral particles and their RNA cargo.

[0221] Preferably, retroviral / lentiviral (e.g., SIV) vectors comprising the modified retroviral / lentiviral (e.g., SIV) RNA sequence vectors of the invention exhibit maintained / increased transgene expression compared to the titer of a corresponding retroviral / lentiviral (e.g., SIV) vector comprising an unmodified retroviral / lentiviral (e.g., SIV) RNA sequence. Retroviral / lentiviral (e.g., SIV) vectors comprising the modified retroviral / lentiviral (e.g., SIV) RNA sequence vectors of the invention exhibit maintained / increased transgene expression and maintained / increased vector integration compared to the titer of a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence. Retroviral / lentiviral (e.g., SIV) vectors comprising the modified retroviral / lentiviral (e.g., SIV) RNA sequence vectors of the invention exhibit maintained / increased transgene expression and maintained / increased vector yield / titer compared to the titer of a corresponding retroviral / lentiviral (e.g., SIV) vector comprising an unmodified retroviral / lentiviral (e.g., SIV) RNA sequence. More preferably, retroviral / lentiviral (e.g., SIV) vectors comprising the modified retroviral / lentiviral (e.g., SIV) RNA sequence vectors of the invention exhibit maintained / increased transgene expression, maintained / increased vector integration, and maintained / increased vector yield / titer compared to the titer of a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence retroviral / lentiviral (e.g., SIV) vector.

[0222] The present invention also provides host cells comprising the retroviral / lentiviral (e.g., SIV) vectors of the present invention. Typically, the host cells are mammalian cells, particularly human cells or cell lines. Non-limiting examples of host cells include HEK293 cells (such as HEK293F or HEK293T cells) and 293T / 17 cells. Commercially available cell lines suitable for virus production are also readily available (as described herein).

[0223] How to generate Also described herein are methods for the production of the retroviral / lentiviral (eg, SIV) vectors of the present invention.

[0224] The present inventors have previously demonstrated that the use of a codon-optimized gal-pol gene from SIV does not negatively impact the production titer of SIV vectors pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxoviruses, and can even result in increased vector titer, as described in PCT / GB2022 / 050524, which is incorporated herein by reference in its entirety.

[0225] The inventors have now shown that retroviral / lentiviral (e.g., SIV) vectors can be produced using modified retroviral / lentiviral (e.g., SIV) RNA sequences that avoid the potential safety risks described herein while (i) maintaining or even increasing transgene expression; (ii) maintaining or even increasing retroviral / lentiviral (e.g., SIV) RNA sequence integration into the host cell genome; and / or (iii) maintaining or even increasing retroviral / lentiviral (e.g., SIV) vector yield. Furthermore, the vector genome plasmids used in the production of the retroviral / lentiviral (e.g., SIV) vectors of the present invention can be combined with the use of codon-optimized gag-pol genes described herein, again maintaining or even increasing vector titer.

[0226] Thus, the present invention provides a method of generating a retroviral / lentiviral (e.g., SIV) vector comprising a modified retroviral / lentiviral (e.g., SIV) RNA sequence described herein, wherein the retroviral / lentiviral (e.g., SIV) is pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus and comprises a promoter and a transgene. Preferably, the retroviral / lentiviral (e.g., SIV) vector is a lentiviral vector, with simian immunodeficiency virus (SIV) vectors being particularly preferred.

[0227] The method of the present invention may be a scalable GMP-compliant method.

[0228] The methods of the present invention typically allow for the production of retroviral / lentiviral (e.g., SIV) vectors comprising modified retroviral / lentiviral (e.g., SIV) RNA sequences that have high levels of transgene expression. Typically, the methods of the present invention produce retroviral / lentiviral (e.g., SIV) vectors comprising modified retroviral / lentiviral (e.g., SIV) RNA sequences described herein that are at least equivalent in terms of transgene expression to retroviral / lentiviral (e.g., SIV) vectors comprising the unmodified retroviral / lentiviral (e.g., SIV) RNA sequence from which the modified retroviral / lentiviral (e.g., SIV) RNA sequence is derived (i.e., the corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence), when produced by the same method.

[0229] As used herein, the term "equivalent transgene expression" can be defined as a modified retroviral / lentiviral (e.g., SIV) RNA sequence that does not significantly reduce transgene expression of a retroviral / lentiviral (e.g., SIV) vector compared to a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence. As a non-limiting example, transgene expression by a retroviral / lentiviral (e.g., SIV) vector comprising a modified retroviral / lentiviral (e.g., SIV) RNA sequence into a host / target cell genome is no more than 2-fold lower, no more than 1.5-fold lower, no more than 1.0-fold lower, no more than 0.5-fold lower, no more than 0.25-fold lower, or less than that by a retroviral / lentiviral (e.g., SIV) vector comprising a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence. The term "equivalent transgene expression" may be defined as transgene expression by a retroviral / lentiviral (e.g., SIV) vector containing a modified retroviral / lentiviral (e.g., SIV) RNA sequence into the host / target cell genome is not statistically altered (e.g., p<0.05, p<0.01) compared to transgene expression by a retroviral / lentiviral (e.g., SIV) vector containing a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence produced by the same method.

[0230] Preferably, transgene expression by a retroviral / lentiviral (e.g., SIV) vector comprising a modified retroviral / lentiviral (e.g., SIV) RNA sequence vector into a host / target cell genome is increased compared to transgene expression by a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence vector produced by the same method. Transgene expression by a retroviral / lentiviral (e.g., SIV) vector comprising a modified retroviral / lentiviral (e.g., SIV) RNA sequence into a host / target cell genome may be at least 1.5-fold, at least 2-fold, or at least 2.5-fold higher than transgene expression by a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence vector produced by the same method.

[0231] The methods of the present invention typically allow for the production of retroviral / lentiviral (e.g., SIV) vectors comprising modified retroviral / lentiviral (e.g., SIV) RNA sequences that have high levels of vector integration into host / target cell genomes. Typically, the methods of the present invention produce retroviral / lentiviral (e.g., SIV) vectors comprising modified retroviral / lentiviral (e.g., SIV) RNA sequences described herein that are at least equivalent in terms of integration into host / target cell genomes compared to retroviral / lentiviral (e.g., SIV) vectors comprising corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequences produced by the same methods.

[0232] As used herein, the term "equivalent integration" can be defined as a modified retroviral / lentiviral (e.g., SIV) RNA sequence that does not significantly reduce integration of the retroviral / lentiviral (e.g., SIV) vector into the host / target cell genome compared to the corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence. As a non-limiting example, integration of the retroviral / lentiviral (e.g., SIV) vector comprising the modified retroviral / lentiviral (e.g., SIV) RNA sequence into the host / target cell genome is no more than 2-fold lower, no more than 1.5-fold lower, no more than 1.0-fold lower, no more than 0.5-fold lower, no more than 0.25-fold lower, or less than that of the retroviral / lentiviral (e.g., SIV) vector comprising the corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence into the host / target cell genome. The term "equivalent integration" may be defined such that integration of a retroviral / lentiviral (e.g., SIV) vector comprising a modified retroviral / lentiviral (e.g., SIV) RNA sequence into the host / target cell genome is statistically unchanged (e.g., p<0.05, p<0.01) compared to integration of a retroviral / lentiviral (e.g., SIV) vector comprising a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence produced by the same method.

[0233] Preferably, integration of the retroviral / lentiviral (e.g., SIV) vector comprising the modified retroviral / lentiviral (e.g., SIV) RNA sequence vector into the host / target cell genome is increased compared to integration of a corresponding retroviral / lentiviral (e.g., SIV) vector comprising an unmodified retroviral / lentiviral (e.g., SIV) RNA sequence produced by the same method. Integration of the retroviral / lentiviral (e.g., SIV) vector comprising the modified retroviral / lentiviral (e.g., SIV) RNA sequence into the host / target cell genome may be at least 1.5-fold, at least 2-fold, or at least 2.5-fold higher than integration of a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence produced by the same method.

[0234] The methods of the present invention typically allow for the production of high titers of purified retroviral / lentiviral (e.g., SIV) vectors comprising modified retroviral / lentiviral (e.g., SIV) RNA sequences. Typically, the methods of the present invention produce titers of retroviral / lentiviral (e.g., SIV) vectors having modified retroviral / lentiviral (e.g., SIV) RNA sequences described herein that are at least equivalent to the titers of retroviral / lentiviral (e.g., SIV) vectors comprising corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequences when produced by the corresponding method.

[0235] As used herein, the term "equivalent potency" can be defined as a modified retroviral / lentiviral (e.g., SIV) RNA sequence that does not significantly reduce the titer of a retroviral / lentiviral (e.g., SIV) vector compared to a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence. As a non-limiting example, the titer of a retroviral / lentiviral (e.g., SIV) vector comprising a modified retroviral / lentiviral (e.g., SIV) RNA sequence is no more than 2-fold lower, no more than 1.5-fold lower, no more than 1.0-fold lower, no more than 0.5-fold lower, no more than 0.25-fold lower, or less than the titer of a retroviral / lentiviral (e.g., SIV) vector comprising a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence. The term "equivalent titer" may be defined such that the titer of a retroviral / lentiviral (e.g., SIV) vector comprising a modified retroviral / lentiviral (e.g., SIV) RNA sequence is not statistically altered (e.g., p<0.05, p<0.01) compared to the titer of a retroviral / lentiviral (e.g., SIV) vector comprising a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence produced by the same method.

[0236] Preferably, the titer of a retroviral / lentiviral (e.g., SIV) vector comprising a modified retroviral / lentiviral (e.g., SIV) RNA sequence vector is increased compared to the titer of a corresponding retroviral / lentiviral (e.g., SIV) vector comprising an unmodified retroviral / lentiviral (e.g., SIV) RNA sequence produced by the same method. The titer of a retroviral / lentiviral (e.g., SIV) vector comprising a modified retroviral / lentiviral (e.g., SIV) RNA sequence may be at least 1.5-fold, at least 2-fold, or at least 2.5-fold higher than the titer of a corresponding retroviral / lentiviral (e.g., SIV) vector comprising an unmodified retroviral / lentiviral (e.g., SIV) RNA sequence produced by the same method.

[0237] Production of retroviral / lentiviral (e.g., SIV) vectors typically uses one or more plasmids that provide the elements necessary for generation of the vector: the Gag-Pol, Rev, F, and HN genomes for the retroviral / lentiviral vector. Multiple elements can be provided on a single plasmid. Preferably, each element is provided on a separate plasmid, so that there are five plasmids, one for each of the Gag-Pol, Rev, F, and HN genomes of the vector.

[0238] Alternatively, a single plasmid may provide the Gag-Pol and Rev elements and may be referred to as the packaging plasmid (pDNA2). The remaining elements (genome, F, and HN) may be provided by separate plasmids (pDNA1, pDNA3a, and pDNA3b, respectively), resulting in four plasmids used for the generation of retroviral / lentiviral (e.g., SIV) vectors according to the invention. In the four-plasmid method, pDNA1, pDNA3a, and pDNA3b may be as described herein in the context of the five-plasmid method.

[0239] In the preferred five-plasmid method of the present invention, the vector genome plasmid encodes all genetic material to be packaged into the final retroviral / lentiviral vector, including the transgene. The vector genome plasmid, sometimes designated "pDNA1" herein, typically contains the transgene and transgene promoter. As described herein, only a portion of the genetic material found in the vector genome plasmid ultimately ends up in the virus, and the exact limits and boundaries of this portion cannot be easily estimated based on the primary sequence of pDNA1. The present invention is the first to elucidate the nucleic acid sequence of a modified RNA sequence in an SIV vector that addresses a number of potential safety risks while providing for (i) transgene expression, (ii) SIV RNA sequence integration, and / or (iii) maintenance or even increase in vector yield.

[0240] The other four plasmids make up the plasmids encoding the Gag-Pol, Rev, F, and HN proteins. These plasmids may be designated "pDNA2a," "pDNA2b," "pDNA3a," and "pDNA3b," respectively.

[0241] Typically, the lentivirus is SIV, such as SIV1, preferably SIV-AGM. The F and HN proteins are derived from a respiratory paramyxovirus, preferably Sendai virus.

[0242] In a specific embodiment relating to CFTR, five plasmids are characterized by Figures 1A-1F, whereby pDNA1 is the pGM830 plasmid of Figure 1A, pDNA2a is the pGM691 plasmid of Figure 1B or the pGM297 plasmid of Figure 1C, pDNA2b is the pGM299 plasmid of Figure 1D, pDNA3a is the pGM301 plasmid of Figure 1E, and pDNA3b is the pGM303 plasmid of Figure 1F, or a variant thereof of any of these plasmids (as described herein). pGM326 (as shown in Figure 1G) is the unmodified version of the vector genome plasmid from which pGM830 is derived.

[0243] When the methods of the invention are used to produce A1AT, the five plasmids may be characterized by Figure 2 (so that plasmid pDNA1 may be pGM407), and all of Figures 1B or 1C and 1D-1F (as described above for specific CFTR embodiments), or variants of any of these plasmids (as described herein).

[0244] When the method of the present invention is used to produce FVIII, the five plasmids may be characterized by one of Figures 3A-3D (so that plasmid pDNA1 may be pGM411, pGM412, pGM413 or pGM414) and all of Figures 1B or 1C and 1D-1F, or variants of any of these plasmids (as described herein).

[0245] The plasmid defined in Figure 1A is represented by SEQ ID NO: 19; the plasmid defined in Figure 1B is represented by SEQ ID NO: 20; the plasmid defined in Figure 1C is represented by SEQ ID NO: 21; the plasmid defined in Figure 1D is represented by SEQ ID NO: 22; the plasmid defined in Figure 1E is represented by SEQ ID NO: 23; the plasmid defined in Figure 1F is represented by SEQ ID NO: 24; the plasmid defined in Figure 1G is represented by SEQ ID NO: 25; the plasmid defined in Figure 2 is represented by SEQ ID NO: 40, and the F / HN-SIV-CMV-HFVIII-V3, F / HN-SIV-hCEF-HFVIII-V3, F / HN-SIV-CMV-HFVIII-N6-co and / or F / HN-SIV-hCEF-HFVIII-N6-co plasmids defined in Figures 3A-3D are represented by SEQ ID NOs: 41-44, respectively. Variants of these plasmids (as defined herein) are also encompassed by the present invention. In particular, variants having at least 90% (such as at least 90, 92, 94, 95, 96, 97, 98, 99, 99.5 or 100%) sequence identity to any one of SEQ ID NOs: 19-25 and 40-44 are encompassed.

[0246] In the five-plasmid method of the present invention, all five plasmids contribute to the formation of the final retroviral / lentiviral (e.g., SIV) vector, but only the vector genome plasmid provides the nucleic acid sequences contained in the retroviral / lentiviral (e.g., SIV) RNA sequence. During the production of a retroviral / lentiviral (e.g., SIV) vector, the vector genome plasmid (pDNA1) provides the enhancer / promoter, Psi, RRE, cPPT, mWPRE, SIN LTR, and SV40 polyA (see Figure 1A), which are important for virus production. Using pGM830 as a non-limiting example of pDNA1, the CMV enhancer / promoter, SV40 polyA, colE1 Ori, and KanR are involved in the production of the retroviral / lentiviral (e.g., SIV) vector of the present invention (e.g., vGM195 or vGM244), but are not found in the final retroviral / lentiviral (e.g., SIV) vector. The RRE, cPPT (central polypurine tract), hCEF, soCFTR2 (transgene), and mWPRE from pGM326 or pGM830 are found in the final retroviral / lentiviral (e.g., SIV) vector. The SIN LTR (long terminal repeat, SIN / IN self-inactivating) and Psi (packaging signal) may be found in the final retroviral / lentiviral (e.g., SIV) vector.

[0247] For other retroviral / lentiviral (e.g., SIV) vectors of the invention, corresponding elements from other vector genome plasmids (pDNA1) are required for production (but are not found in the final vector) or are present in the final retroviral / lentiviral (e.g., SIV) vector.

[0248] The F and HN proteins (preferably Sendai F and HN proteins) from pDNA3a and pDNA3b are important for infection of target cells by the final retroviral / lentiviral (e.g., SIV) vector, i.e., for entry of the patient's epithelial cells (typically lung or nasal cells as described herein). The products of the pDNA2a and pDNA2b plasmids are important for viral transduction, i.e., for insertion of retroviral / lentiviral (e.g., SIV) DNA into the host genome. The promoter, regulatory elements (such as WPRE), and transgene are important for transgene expression in the target cells.

[0249] The methods of the present invention may comprise or consist of the following steps: (a) growing cells in suspension; (b) transfecting the cells with one or more plasmids; (c) adding a nuclease; (d) harvesting the lentivirus (e.g., SIV); (e) adding trypsin; and (f) purifying the lentivirus (e.g., SIV).

[0250] This method may use the four- or five-plasmid system described herein. Thus, for the preferred five-plasmid method, one or more plasmids may comprise or consist of a vector genome plasmid, pDNA1; a gagpol plasmid (e.g., a codon-optimized gagpol plasmid), pDNA2a; a Rev plasmid, pDNA2b; a fusion (F) protein plasmid, pDNA3a; and a hemagglutinin-neuraminidase (HN) plasmid, pDNA3b. pDNA1 may be pGM830. pDNA2a may be pGM297 or pGM691, preferably pGM691. pDNA2b may be pGM299. pDNA3a may be pGM301. pDNA3b may be pGM303. Any combination of pDNA1, pDNA2a, pDNA2b, pDNA3a, and pDNA3b may be used. Preferably, pDNA1 is pGM830; pDNA2a is pGM691; pDNA2b is pGM299; pDNA3a is pGM301; and pDNA3b is pGM303.

[0251] Any appropriate ratio of vector genome plasmid:gagpol plasmid:Rev plasmid:F plasmid:HN plasmid may be used to further optimize (increase) the resulting retroviral / lentiviral (e.g., SIV) titer. As a non-limiting example, the ratio of vector genome plasmid:gagpol plasmid:Rev plasmid:F plasmid:HN plasmid may be in the range of 10:40:4, 20:3, 12:3, 12:3, 12:12, typically 15:20:7, 11:4, 8:4, 8:4, 8:4, 8, e.g., approximately 18:22:7, 11:4, 8:4, 8:4, 8, e.g., approximately 19:21:8, 10:5, 7:5, 7:5, 7:5, 7:7. Preferably, the ratio of vector genome plasmid:gagpol plasmid:Rev plasmid:F plasmid:HN plasmid is approximately 20:9:6:6:6.

[0252] Steps (a) through (f) of the method are typically performed sequentially, beginning with step (a) and continuing through step (f). The method may include one or more additional steps, such as an additional purification step, buffer exchange, concentration of the purified retroviral / lentiviral (e.g., SIV) vector, and / or formulation of the purified (or concentrated) retroviral / lentiviral (e.g., SIV) vector. Each of the steps may include one or more substeps. For example, the harvesting step may include one or more steps or substeps, and / or the purification may include one or more steps or substeps.

[0253] Any suitable cell type may be transfected with one or more plasmids (e.g., the five plasmids described herein) to produce the retroviral / lentiviral (e.g., SIV) vectors of the present invention. Typically, mammalian cells, particularly human cell lines, are used. Non-limiting examples of cells suitable for use in the methods of the present invention are HEK293 cells (such as HEK293F or HEK293T cells) and 293T / 17 cells. Commercially available cell lines suitable for virus production are also readily available (e.g., Gibco Viral Production Cells from ThermoFisher Scientific - Catalog No. A35347).

[0254] The cells may be grown in animal component-free media, including serum-free media. The cells may be grown in media containing human components. The cells may be grown in defined media that contain or consist of synthetically produced components.

[0255] Any suitable transfection means may be used in accordance with the present invention. The selection of a suitable transfection means is within the routine practice of one skilled in the art. As a non-limiting example, transfection may be performed by the use of PEIPro™, Lipofectamine 2000™, or Lipofectamine 3000™.

[0256] Any suitable nuclease may be used in accordance with the present invention. The selection of a suitable nuclease is within the routine practice of one skilled in the art. Typically, the nuclease is an endonuclease. As a non-limiting example, the nuclease may be Benzonase® or Denarase®. The addition of the nuclease may be at a pre-harvesting stage, a post-harvesting stage, or during the harvesting step.

[0257] The gag-pol gene used in generating the retroviral / lentiviral (e.g., SIV) vectors of the present invention may be codon-optimized. Thus, the gag-pol gene in the pDNA2a plasmid may be codon-optimized. As a non-limiting example, the codon-optimized gag-pol gene may comprise or consist of the nucleic acid sequence of SEQ ID NO: 17, or a variant thereof (as defined herein). In particular, the codon-optimized gag-pol gene of the present invention may comprise or consist of a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 17, preferably at least 95% identity to SEQ ID NO: 17. The codon-optimized gag-pol gene may consist of the nucleic acid sequence of SEQ ID NO: 17. A preferred pDNA2a vector, pGM691, contains the codon-optimized gag-pol gene of SEQ ID NO: 17.

[0258] Gag-pol genes (e.g., SIV gag-pol genes), including codon-optimized gag-pol genes, are typically operably linked to a promoter to promote expression of the gag-pol protein. Any suitable promoter may be used, including those described herein in the context of promoters for transgenes. Preferably, the promoter is a CAG promoter when used in the exemplified pGM691 plasmid. An exemplary CAG promoter is set forth in SEQ ID NO: 45. The codon-optimized gag-pol gene of SEQ ID NO: 17 contains translation slippage and therefore does not form a single conventional open reading frame.

[0259] Codon-optimized gag-pol genes (or nucleic acids comprising or consisting of them) and plasmids comprising the genes or nucleic acids are advantageous in the production of retroviral / lentiviral (e.g., SIV) vectors using the methods of the present invention because they enable the production of high-titer F / HN retroviral / lentiviral (e.g., SIV) vectors. Typically, the codon-optimized gag-pol genes (or nucleic acids comprising or consisting of them) and plasmids comprising the genes or nucleic acids can be used to produce retroviral / lentiviral (e.g., SIV) vector titers that are at least equivalent to the titers of retroviral / lentiviral (e.g., SIV) vectors produced by corresponding methods that do not use a codon-optimized gag-pol gene, as described herein. Therefore, the use of a codon-optimized gag-pol gene can be combined with modified retroviral / lentiviral (e.g., SIV) RNA sequences to further maintain or increase vector titers.

[0260] The codon-optimized gag-pol gene is further disclosed in PCT / GB2022 / 050524, which is incorporated herein by reference in its entirety.

[0261] The present invention also provides retroviral / lentiviral (eg, SIV) vectors obtainable by the methods of the present invention.

[0262] Typically, retroviral / lentiviral (e.g., SIV) vectors obtainable by the methods of the present invention are produced at high titers, as described herein. Titers may be measured in terms of transducing units, as defined herein. As described herein, the methods of the present invention typically produce retroviral / lentiviral (e.g., SIV) vectors comprising modified retroviral / lentiviral (e.g., SIV) RNA sequences at titers comparable to or higher than those of corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA vectors and methods that do not use a codon-optimized gag-pol gene.

[0263] Thus, retroviral / lentiviral (e.g., SIV) vectors of the invention, including those obtainable by the methods of the invention, optionally contain at least about 2.5 x 10 6 TU / mL, at least approximately 3.0 × 10 6 TU / mL, at least approximately 3.1 × 10 6 TU / mL, at least approximately 3.2 × 10 6 TU / mL, at least approximately 3.3 × 10 6 TU / mL, at least approximately 3.4 × 10 6 TU / mL, at least approximately 3.5 × 10 6 TU / mL, at least approximately 3.6 × 10 6 TU / mL, at least approximately 3.7 × 10 6 TU / mL, at least approximately 3.8 × 10 6 TU / mL, at least approximately 3.9 × 10 6 TU / mL, at least approximately 4.0 × 10 6 Preferably, the retroviral / lentiviral (e.g., SIV) vector has a titer of at least about 3.0 x 10 TU / mL. 6TU / mL, or at least about 3.5 x 10 6 It is produced in titers of TU / mL.

[0264] The production of high titers of retroviral / lentiviral (e.g., SIV) vectors may impart other desirable properties to the resulting vector product. For example, without being bound by theory, it is believed that production at high titers without the need for significant concentration by methods such as TFF results in a higher quality vector product than retroviral / lentiviral (e.g., SIV) vectors produced by corresponding methods that do not use codon-optimized gag-pol genes (and optionally, modified vector genome plasmids), because the vectors are exposed to less shear forces that can damage viral particles and their RNA cargo.

[0265] Typically, the gag-pol gene (e.g., a codon-optimized gag-pol gene) used matches the retroviral / lentiviral vector to be generated. As a non-limiting example, if the lentiviral vector is an HIV vector, the codon-optimized gag-pol gene used is the HIV gag-pol gene. As a non-limiting example, if the lentiviral vector is an SIV vector, the codon-optimized gag-pol gene used is the SIV gag-pol gene.

[0266] Preferably, the codon-optimized gag-pol gene used is the SIV gag-pol gene.

[0267] As described herein, the retroviral / lentiviral (e.g., SIV) vector of the present invention comprises a modified retroviral / lentiviral (e.g., SIV) RNA sequence, which is typically modified to reduce the number of retroviral / lentiviral (e.g., SIV) ORFs. Thus, the vector genome plasmid used in the production of the retroviral / lentiviral (e.g., SIV) vector of the present invention may be modified to reduce the number of retroviral / lentiviral (e.g., SIV) ORFs. Any disclosure herein regarding the modification of retroviral / lentiviral (e.g., SIV) RNA sequences, including modifications to reduce the number of retroviral / lentiviral (e.g., SIV) ORFs in the retroviral / lentiviral (e.g., SIV) RNA sequence, applies equally and unconditionally to the vector genome plasmid (pDNA1) described herein, which can be used in the production of the retroviral / lentiviral (e.g., SIV) vector of the present invention.

[0268] As used herein, the term "trypsin" refers to both trypsin and its equivalents. Equivalent enzymes are those that have the same or essentially the same cleavage specificity as trypsin. Trypsin cleavage activity can be defined as cleavage C-terminal to arginine or lysine residues, typically exclusively C-terminal to arginine or lysine residues. Trypsin activity may preferably be provided by an animal-origin-free recombinant enzyme, such as TrypLE Select™. Addition of trypsin may occur at a pre-harvest or post-harvest stage, or during the harvesting step.

[0269] Any suitable purification means may be used to purify retroviral / lentiviral (e.g., SIV) vectors. Non-limiting examples of suitable purification steps include depth / end filtration, tangential flow filtration (TFF), and chromatography. A purification step typically includes at least one chromatography step. Non-limiting examples of chromatography steps that can be used in accordance with the present invention include mixed-mode size exclusion chromatography (SEC) and / or anion exchange chromatography. Elution may be performed with or without, preferably without, the use of a salt gradient.

[0270] This method may be used to generate retroviral / lentiviral (e.g., SIV) vectors of the invention, such as those comprising the CFTR, A1AT, and / or FVIII genes described herein. Alternatively, the retroviral / lentiviral (e.g., SIV) vectors of the invention comprise any of the above-listed genes or genes encoding the above-listed proteins.

[0271] Retroviral / lentiviral (e.g., SIV) vectors of the present invention are provided using methods that may employ any combination of one or more of the specific plasmid constructs provided by Figures 1A-1F, 2, and / or 3A-3D. In particular, the plasmid constructs of Figures 1B and 1D-1F are preferably used in combination with the plasmids of Figure 1A, 2, or 3A-3D, with the plasmid of Figure 1A being particularly preferred.

[0272] The present invention also provides a method for increasing retroviral / lentiviral (e.g., SIV) vector titer, comprising the use of a modified retroviral / lentiviral (e.g., SIV) RNA sequence described herein, or a vector genome plasmid from which such a modified retroviral / lentiviral (e.g., SIV) RNA sequence is derived. This method may be combined with the use of a codon-optimized gag-pol gene (or a nucleic acid comprising or consisting of the same), a plasmid comprising said gene or nucleic acid described herein, to further increase retroviral / lentiviral (e.g., SIV) vector titer. The methods of increasing retroviral / lentiviral (e.g., SIV) vector titer according to the present invention may increase titer by at least 1.5-fold, at least 2-fold, or at least 2.5-fold or more compared to corresponding methods using a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence or vector genome plasmid from which the corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence is derived, and may also use a non-codon-optimized version of the gag-pol gene (or a nucleic acid comprising or consisting of the same), or a plasmid or host cell comprising the non-codon-optimized gag-pol gene or nucleic acid. Alternatively, methods of increasing retroviral / lentiviral (e.g., SIV) titer according to the present invention may increase titer by at least about 25%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, or more, compared to corresponding methods using a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence or vector genome plasmid from which the corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence is derived, and may also use a non-codon-optimized version of the gag-pol gene (or a nucleic acid comprising or consisting of the same), or a plasmid comprising said non-codon-optimized gene or nucleic acid.Preferably, the method of increasing retroviral / lentiviral (e.g., SIV) vector titer according to the present invention may increase titer by (a) at least 1.5-fold or at least 2-fold; and / or (b) at least about 25%, more preferably at least about 50%, and even more preferably at least about 100%. Typically, the corresponding method is identical to the method of the present invention, except for the use of a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence or vector genome plasmid from which the corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence is derived, and, optionally, a codon-optimized gag-pol gene (or a nucleic acid comprising or consisting thereof), or a plasmid comprising said gene or nucleic acid. All disclosures herein relating to methods of generating retroviral / lentiviral (e.g., SIV) vectors apply equally and unconditionally to the method of increasing retroviral / lentiviral (e.g., SIV) titer according to the present invention.

[0273] The present invention also provides the use of a modified retroviral / lentiviral (e.g., SIV) RNA sequence of the present invention (or a vector genome plasmid from which said modified retroviral / lentiviral (e.g., SIV) RNA sequence is derived) to increase the titer of a retroviral / lentiviral (e.g., SIV) vector. This use may be combined with the use of a codon-optimized gag-pol gene (or a nucleic acid comprising or consisting thereof), a plasmid comprising said gene or nucleic acid as described herein, to further increase retroviral / lentiviral (e.g., SIV) vector titer. Said use may increase retroviral / lentiviral (e.g., SIV) vector titer by at least 1.5-fold, at least 2-fold, or at least 2.5-fold, or more, compared to use of a plasmid comprising the modified retroviral / lentiviral (e.g., SIV) RNA sequence of the invention (or the vector genome plasmid from which said modified retroviral / lentiviral (e.g., SIV) RNA sequence is derived) and, optionally, the corresponding non-codon-optimized version of the gag-pol gene (or nucleic acid comprising or consisting thereof), or said non-codon-optimized gene or nucleic acid. Alternatively, the use may increase retroviral / lentiviral (e.g., SIV) titer by at least about 25%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, or more, compared to use of a plasmid comprising a modified retroviral / lentiviral (e.g., SIV) RNA sequence of the invention (or the vector genome plasmid from which the modified retroviral / lentiviral (e.g., SIV) RNA sequence is derived) and, optionally, a corresponding non-codon-optimized version of the gag-pol gene (or a nucleic acid comprising or consisting thereof), or said non-codon-optimized gene or nucleic acid.Preferably, the use increases the retroviral / lentiviral (e.g., SIV) titer by (a) at least 1.5-fold or at least 2-fold; and / or (b) at least about 25%, more preferably at least about 50%, and even more preferably at least about 100%. Typically, the corresponding use is identical to the method of the present invention, except for the use of a modified retroviral / lentiviral (e.g., SIV) RNA sequence of the present invention (or a vector genome plasmid from which the modified retroviral / lentiviral (e.g., SIV) RNA sequence is derived), and optionally a codon-optimized gag-pol gene (or a nucleic acid comprising or consisting of the same), a plasmid comprising said gene or nucleic acid. All disclosures herein relating to methods of generating retroviral / lentiviral (e.g., SIV) vectors apply equally and unconditionally to the use of the modified retroviral / lentiviral (e.g., SIV) RNA sequences of the invention (or the vector genome plasmid from which said modified retroviral / lentiviral (e.g., SIV) RNA sequences are derived), and optionally the codon-optimized gag-pol gene (or a nucleic acid comprising or consisting thereof), plasmids comprising said gene or nucleic acid, to increase the titer of retroviral / lentiviral (e.g., SIV) vectors in accordance with the invention.

[0274] The use of a codon-optimized gag-pol gene in combination with a modified retroviral / lentiviral (e.g., SIV) RNA sequence of the invention, or a vector genome plasmid from which the modified retroviral / lentiviral (e.g., SIV) RNA sequence is derived, may provide additional advantages in terms of safety and / or vector titer. Thus, the increased vector yields described herein may be achieved using a modified retroviral / lentiviral (e.g., SIV) RNA sequence of the invention (or a vector genome plasmid from which the modified retroviral / lentiviral (e.g., SIV) RNA sequence is derived) in combination with a codon-optimized gag-pol gene. Any and all disclosures herein relating to increasing vector titer in the context of methods using the modified retroviral / lentiviral (e.g., SIV) RNA sequences of the invention (or the vector genome plasmid from which said modified retroviral / lentiviral (e.g., SIV) RNA sequences are derived) apply equally and unconditionally to methods using the modified retroviral / lentiviral (e.g., SIV) RNA sequences of the invention (or the vector genome plasmid from which said modified retroviral / lentiviral (e.g., SIV) RNA sequences are derived) in combination with a codon-optimized gag-pol gene, and to vectors produced by such methods.

[0275] therapeutic symptoms The retroviral / lentiviral (e.g., SIV) vectors of the present invention enable higher and sustained gene expression through efficient gene transfer while also reducing the risk of side effects caused by the expression of retroviral ORFs, such as upstream ORFs. The F / HN-pseudotyped retroviral / lentiviral (e.g., SIV) vectors of the present invention are capable of (i) airway transduction without disruption of epithelial integrity; (ii) sustained gene expression; (iii) lack of chronic toxicity; and (iv) efficient repeated administration. Long-term / sustained stable gene expression, preferably at therapeutically effective levels, can be achieved using repeated doses of the vectors of the present invention. Alternatively, a single dose may be used to achieve the desired long-term expression.

[0276] Therefore, the retroviral / lentiviral (e.g., SIV) vectors of the present invention can be advantageously used in gene therapy. For example, the efficient airway cell uptake properties of the retroviral / lentiviral (e.g., SIV) vectors of the present invention make them highly suitable for treating respiratory airway diseases. The retroviral / lentiviral (e.g., SIV) vectors of the present invention can also be used in gene therapy methods to promote the secretion of therapeutic proteins. As a further example, the present invention provides for the secretion of therapeutic proteins into the lumen of the respiratory airway or into the circulatory system. Thus, administration of the retroviral / lentiviral (e.g., SIV) vectors of the present invention and their uptake by airway cells may enable the use of the lungs (or nose or airways) as a "factory" for producing therapeutic proteins that can then be secreted, enter the systemic circulation at therapeutic levels, and migrate to desired cells / tissues, where they can elicit a therapeutic effect. In contrast to intracellular or membrane proteins, the production of such secreted proteins does not rely on specific disease target cells to be transduced, which is a significant advantage, achieving high levels of protein expression. Therefore, diseases other than respiratory tract diseases, such as cardiovascular diseases and blood disorders, particularly blood clotting deficiencies, can also be treated by the retroviral / lentiviral (eg, SIV) vectors of the present invention.

[0277] The retroviral / lentiviral (e.g., SIV) vector of the present invention can effectively treat disease by providing a transgene for disease correction. For example, inserting a functional copy of the CFTR gene can ameliorate or prevent lung disease in CF patients, regardless of underlying mutations. Therefore, the retroviral / lentiviral (e.g., SIV) vector of the present invention can be used to treat cystic fibrosis (CF) by gene therapy, typically using the CFTR transgene described herein.

[0278] As another example, the retroviral / lentiviral (e.g., SIV) vectors of the present invention may be used to treat A1AT deficiency, typically by gene therapy using the alpha-1 antitrypsin (A1AT) transgene described herein. A1AT is a secreted antiprotease primarily produced in the liver and then transported to the lungs, with smaller amounts also produced in the lungs themselves. The primary function of A1AT is to bind to and neutralize / inhibit neutrophil elastase. Gene therapy using A1AT according to the present invention is relevant not only to patients with A1AT deficiency but also to other lung diseases, such as CF or chronic obstructive pulmonary disease (COPD), and offers the opportunity to overcome some of the problems encountered with conventional enzyme replacement therapy (in which A1AT is isolated from human blood and administered intravenously weekly), providing stable and long-lasting expression in target tissues (lung / nasal epithelium), ease of administration, and unlimited availability.

[0279] Transduction with retroviral / lentiviral (e.g., SIV) vectors of the present invention can result in secretion of recombinant proteins into the lumen of the lung and into the circulation. One benefit of this is that the therapeutic protein reaches the interstitium. Thus, A1AT gene therapy may also be beneficial in other disease conditions, non-limiting examples of which include type 1 and type 2 diabetes, acute myocardial infarction, ischemic heart disease, rheumatoid arthritis, inflammatory bowel disease, transplant rejection, graft-versus-host (GvH) disease, multiple sclerosis, liver disease, cirrhosis, vasculitis, and infectious diseases, such as bacterial and / or viral infections.

[0280] A1AT has numerous other anti-inflammatory and tissue protective effects, for example, in preclinical models of diabetes, graft-versus-host disease, and inflammatory bowel disease. Production of A1AT in the lung and / or nose after transduction according to the present invention may therefore be more widely applicable, including to these conditions.

[0281] Other examples of diseases that can be treated using secreted protein gene therapy according to the present invention include cardiovascular diseases and blood disorders, in particular blood clotting deficiencies such as hemophilia (A, B or C), von Willebrand's disease and factor VII deficiency.

[0282] Other examples of diseases or disorders to be treated include Primary Ciliary Dyskinesia (PCD), Acute Lung Injury, Surfactant Protein B (SFTB) Deficiency, Pulmonary Alveolar Proteinosis (PAP), Chronic Obstructive Pulmonary Disease (COPD), and / or inflammatory, infectious, immune, or metabolic conditions, such as lysosomal storage diseases.

[0283] Therefore, the present invention provides a method for treating a disease, comprising administering a retroviral / lentiviral (e.g., SIV) vector of the present invention to a subject. Typically, the retroviral / lentiviral (e.g., SIV) vector is produced using the method of the present invention. Any disease described herein can be treated according to the present invention. In particular, the present invention provides a method for treating a lung disease using the retroviral / lentiviral (e.g., SIV) vector of the present invention. The disease to be treated may be a chronic disease. Preferably, a method for treating CF is provided.

[0284] The present invention also provides the retroviral / lentiviral (e.g., SIV) vector described herein for use in a method for treating a disease. Typically, the retroviral / lentiviral (e.g., SIV) vector is produced using the method of the present disclosure. Any disease described herein can be treated according to the present invention. In particular, the present invention provides the retroviral / lentiviral (e.g., SIV) vector of the present invention for use in a method for treating a lung disease. The disease to be treated may be a chronic disease. Preferably, a retroviral / lentiviral (e.g., SIV) vector is provided for use in the treatment of CF.

[0285] The present invention also provides the use of the retroviral / lentiviral (e.g., SIV) vector described herein in the manufacture of a medicament for use in a method for treating a disease. Typically, the retroviral / lentiviral (e.g., SIV) vector is produced using the method of the present disclosure. Any disease described herein can be treated according to the present invention. In particular, the present invention provides the use of the retroviral / lentiviral (e.g., SIV) vector of the present invention in the manufacture of a medicament for use in a method for treating a lung disease. The disease to be treated may be a chronic disease. Preferably, the use of the retroviral / lentiviral (e.g., SIV) vector in the manufacture of a medicament for use in a method for treating CF is provided.

[0286] Formulation and Administration The retroviral / lentiviral (e.g., SIV) vectors of the present invention may be administered at any dosage appropriate to achieve the desired therapeutic effect. Appropriate dosages can be determined by clinicians or other physicians within the normal course of their work using standard techniques. Non-limiting examples of suitable dosages include 1 x 10 8 Transduction unit (TU), 1 x 10 9 TU, 1×10 10 TU, 1×10 11 TU or higher.

[0287] The present invention also provides a composition comprising the retroviral / lentiviral (e.g., SIV) vector described above and a pharmaceutically acceptable carrier. Non-limiting examples of pharmaceutically acceptable carriers include water, saline, and phosphate-buffered saline. In some embodiments, however, the composition is in lyophilized form, which may contain a stabilizer, such as bovine serum albumin (BSA). In some embodiments, it may be desirable to formulate the composition with a preservative, such as thiomersal or sodium azide, to facilitate long-term storage.

[0288] The retroviral / lentiviral (e.g., SIV) vectors of the present invention may be administered by any suitable route. It may be desirable to direct the compositions of the present invention (as described above) to the respiratory system of a subject. Efficient delivery of the therapeutic / prophylactic composition or medicament to the site of infection in the respiratory tract may be achieved by oral or intranasal administration, for example, as an aerosol (e.g., a nasal spray), or by catheter. Typically, the retroviral / lentiviral (e.g., SIV) vectors of the present invention are stable in clinically relevant nebulizers, inhalation devices (including metered-dose inhalers), catheters, aerosols, and the like. Typically, therefore, the retroviral / lentiviral (e.g., SIV) vectors of the present invention are formulated for pulmonary administration by any suitable means; for example, they may be formulated for intratracheal administration, intranasal administration, aerosol delivery, or direct injection or delivery to the lungs (e.g., delivered by catheter). Other modes of delivery, such as intravenous delivery, are also encompassed by the present invention.

[0289] In some embodiments, the nose is a preferred production site for therapeutic proteins using the retroviral / lentiviral (e.g., SIV) vectors of the present invention for at least one of the following reasons: (i) extracellular barriers, such as inflammatory cells and sputum, are less prominent in the nose; (ii) vector administration is easier; (iii) smaller amounts of vector are required; and (iv) ethical considerations. Therefore, transduction of nasal epithelial cells with the retroviral / lentiviral (e.g., SIV) vectors of the present invention can result in efficient (high-level) and long-lasting expression of the desired therapeutic transgene. Therefore, nasal administration of the retroviral / lentiviral (e.g., SIV) vectors of the present invention may be preferred.

[0290] Formulations for intranasal administration may be in the form of nasal drops or nasal sprays. Nasal formulations may contain droplets having an approximate diameter in the range of 100 to 5000 μm, e.g., 500 to 4000 μm, 1000 to 3000 μm, or 100 to 1000 μm. Alternatively, in terms of volume, the droplets may be in the range of about 0.001 to 100 μl, e.g., 0.1 to 50 μl, or 1.0 to 25 μl, or e.g., 0.001 to 1 μl.

[0291] Aerosol formulations may take the form of a powder, suspension, or solution. The size of the aerosol particles is related to the delivery capacity of the aerosol. Smaller particles may travel further down the respiratory tract toward the alveoli than larger particles. In one embodiment, the aerosol particles have a diameter distribution to facilitate delivery along the entire length of the bronchi, bronchioles, and alveoli. Alternatively, the particle size distribution may be selected to target a specific section of the respiratory tract, such as the alveoli. In the case of aerosol delivery of pharmaceuticals, the particles may have a diameter within the approximate range of 0.1 to 50 μm, preferably 1 to 25 μm, and more preferably 1 to 5 μm.

[0292] The aerosol particles may be for delivery using a nebulizer (e.g., via the mouth) or a nasal spray. The aerosol formulation may optionally contain a propellant and / or a surfactant.

[0293] The formulation of pharmaceutical aerosols is routine for those skilled in the art; see, for example, Sciarra, J. in Remington's Pharmaceutical Sciences (supra). The medicament may be formulated as a solution aerosol, a dry powder dispersion or suspension aerosol, an emulsion, or a semisolid preparation. The aerosol may be delivered using any propellant system known to those skilled in the art. The aerosol may be applied to the upper respiratory tract, the lower respiratory tract, or both, for example, by nasal inhalation. The portion of the lung to which the medicament is delivered may be determined by the disorder. Compositions containing the vectors of the present invention may also contain a humectant, particularly when intranasal delivery is used. This may help reduce or prevent drying of the mucous membranes and prevent membrane irritation. Suitable humectants include, for example, sorbitol, mineral oil, vegetable oil, and glycerol; soothing agents; membrane conditioners; sweeteners; and combinations thereof. The composition may also contain a surfactant. Suitable surfactants include nonionic, anionic, and cationic surfactants. Examples of surfactants that can be used include polyoxyethylene derivatives of fatty acid partial esters of sorbitol anhydride, such as Tween 80, polyoxyl 40 stearate, polyoxyethylene 50 stearate, fusieates, bile salts, and octoxynol.

[0294] In some cases, subsequent administrations of retroviral / lentiviral (e.g., SIV) vectors may be administered after the initial administration. Administration may be, for example, at least one week, two weeks, one month, two months, six months, one year, or later after the initial administration. In some instances, the retroviral / lentiviral (e.g., SIV) vectors of the present invention may be administered at least once a week, once every two weeks, once a month, every two months, every six months, every year, or at longer intervals. Preferably, administration is every six months, more preferably every year. The retroviral / lentiviral (e.g., SIV) vector may be administered at indicated intervals, for example, when the effectiveness of previous administrations is diminishing.

[0295] Any two or more retroviral / lentiviral (e.g., SIV) vectors of the present invention may be administered separately, sequentially, or simultaneously. Thus, two retroviral / lentiviral (e.g., SIV) vectors or three or more retroviral / lentiviral (e.g., SIV) vectors, where at least one retroviral / lentiviral (e.g., SIV) vector is a retroviral / lentiviral (e.g., SIV) vector of the present invention, may be administered separately, simultaneously, or sequentially, and particularly, two or more retroviral / lentiviral (e.g., SIV) vectors of the present invention may be administered in such a manner. The two may be administered in the same or different compositions. In a preferred embodiment, the two retroviral / lentiviral (e.g., SIV) vectors may be delivered in the same composition.

[0296] sequence homology Any of a variety of sequence alignment methods can be used to determine percent identity, including, but not limited to, global methods, local methods, and hybrid methods, such as segment approach methods. Protocols for determining percent identity are routine procedures within the skill of one in the art. Global methods align the sequence from the beginning to the end of the molecule and determine the best alignment by adding up the scores of individual residue pairs and imposing gap penalties. Non-limiting methods include, for example, CLUSTAL W, see e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences.Non-limiting methods include, for example: Match-Box, see e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbs sampling, see e.g., C.E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993); Align-M, see e.g., Ivo Van Walle et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004).

[0297] Percent sequence identity is therefore determined by conventional methods. See, e.g., Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment score using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.), as shown below (amino acids are indicated by standard single-letter codes):

[0298] The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, the percent identity can be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids multiplied by 100. The calculation of percent sequence identity may also take into account the number of gaps that need to be introduced to optimize the alignment of two or more sequences, and the length of each gap. Sequence comparison and percent identity determination between two or more sequences can be performed using specific mathematical algorithms familiar to those skilled in the art, such as BLAST.

[0299] Alignment scores for determining sequence identity ARNDCQEGHILKMFPSTWYV A4 R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4

[0300] The percent identity is then calculated as follows: Total number of identical matches _______________________________×100 [length of the longer sequence + number of gaps introduced in the longer sequence to align the two sequences]

[0301] Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions, or additions. These changes are preferably of a minor nature, such as conservative amino acid substitutions (as described herein) and other substitutions that do not significantly affect the folding or activity of the polypeptide; typically small deletions of from 1 to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.

[0302] In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) may be substituted for amino acid residues in the polypeptides of the invention. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues in the polypeptides. The polypeptides of the invention may also include non-naturally occurring amino acid residues.

[0303] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods for incorporating non-naturally occurring amino acid residues into proteins are known in the art. For example, an in vitro system can be used in which nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylation of tRNAs are known in the art. Transcription and translation of the nonsense mutation-containing plasmid is carried out in a cell-free system containing an E. coli S30 extract and commercially available enzymes and other reagents. The protein is purified by chromatography. See, e.g., Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993. In the second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996).In the third method, E. coli cells are cultured in the absence of the natural amino acid to be replaced (e.g., phenylalanine) and in the presence of a desired unnatural amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The unnatural amino acid is incorporated into the polypeptide in place of its natural counterpart. See Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to unnatural species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the scope of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0304] A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may substitute for polypeptide amino acid residues in the polypeptides of the invention.

[0305] Essential amino acids in the polypeptides of the invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244:1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of the structure, determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutations of amino acids at putative contact sites. See, e.g., de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identity of essential amino acids can also be inferred from analysis of homology with related components of the polypeptides of the invention (e.g., translocation or protease components).

[0306] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting functional polypeptides, and then sequencing the mutagenized polypeptides to determine the spectrum of permissible substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0307] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting functional polypeptides, and then sequencing the mutagenized polypeptides to determine the spectrum of permissible substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0308] [Example] The present invention will now be described with reference to the following examples. These are not intended to limit the scope of the invention, and those skilled in the art will recognize that suitable equivalents may be used within the scope of the invention. As such, the examples can be considered as constituent parts of the invention, and the individual aspects described therein can be considered to be disclosed independently or in any combination. [Example]

[0309] Modifications of the vector genome plasmid, including reducing the number of intact SIV ORFs within the vector genome plasmid, maintain or even increase vector yield. The inventors reviewed the sequences of the constructed plasmids and identified several regions of interest within the original vector genome plasmid pGM326. In particular, the pGM326 partial Gag RRE cPPT hCEF region contains: ·77 initiation codon (ATG); 32 ORFs ≥ 10 amino acids in length Two large ORFs in the 5' to 3' direction 189 amino acids from the 5'-most ATG in the vector genome (Gag / RRE fusion), encoding parts of the p17 matrix and p24 capsid 〇 250 amino acids from ATG within the RRE (RRE / cPPT / hCEF fusion)

[0310] In particular, 14 ATG start codons were identified in the partial Gag / RRE region of the pGM326 genomic plasmid that result in ORFs longer than 10 amino acids. These are illustrated in Figure 4. Circular ATGs are those that have a strong Kozak sequence and are in frame with Gag or Env.

[0311] Therefore, we designed a modified version of the pGM326 plasmid with a combination of additional modifications intended to reduce the number of intact SIV ORFs (specifically removing these two large ORFs) for improved safety. Modifications are made to the hCEF promoter and the two large ORFs upstream of the CFTR transgene (soCFTR2). The changes made were as follows: [Table 2] fsATG = frameshift ATG; mtATG = ATG with a point mutation (ATG disruption); mtSTOP = mutated ATG-> stop codon (introduced)

[0312] Approach 1 created frameshift mutations at ATG codons (fsATG) 1, 2, 3, and 5 in the SIV-CFTR partial Gag region. Approach 2 created frameshift mutations at ATG codons 1 and 3 in the SIV-CFTR partial Gag region. Approach 3 created point mutations at ATG codons (mtATG) 1 and 3 in the SIV-CFTR partial Gag region. Approach 4 created a stop codon mutation at codon 6 in the SIV-CFTR partial Gag region and a point mutation at codon 3 in the partial Gag region. Approach 5 created frameshift mutations at ATG codons 1, 2, 3, and 5 and point mutations at codons 7, 12, and 13 in the SIV-CFTR partial Gag / RRE region. Approach 6 created a mutation of codon 6 of the SIV-CFTR partial Gag region to a stop codon, as well as point mutations to ATG codons 3, 7, 12, and 13 across the SIV-CFTR partial Gag / RRE region. Approach 5 generated the vector genome plasmid pGM830 shown in Figure 1A, which has the sequence of SEQ ID NO:19.

[0313] Each new vector genome plasmid was evaluated for functionality by two rounds of transient lentiviral vector (LV) generation, involving transfection of the tested plasmids with SIV GagPol, SIV Rev, SeV Fct4, ​​and SIVct+SeV HN plasmids into A459 cells in a 12 mL volume in an Ambr® 15 bioreactor system. After LV generation, the vector product was activated, filtered through a 0.45 μm filter, and stored at -80°C. After thawing, the activated material was diluted 1:50 and transduced into A459 cells. The resulting LV titer was quantified using CFTR FACS.

[0314] As shown in Figure 5, several modified vector genome plasmids resulted in observable increases in LV titers compared to the unmodified pGM326 vector genome plasmid. The pGM830 vector genome plasmid produced LV titers greater than 1.0 x 10 for unmodified pGM326. 6 Compared with TU / mL, the highest LV titer (6.5 × 10 6 TU / mL).

[0315] Comparison of vector titers using either pGM326 and the modified vector genome plasmid in an otherwise identical production protocol demonstrated that use of the modified vector genome plasmid gave titers at least equivalent to pGM326, indicating that an improved safety profile could be achieved without adversely affecting titer. [Example]

[0316] Modifications of the vector genome plasmid, including reducing the number of intact SIV ORFs within the vector genome plasmid, maintain or even increase vector integration. The LV production of Example 1 was repeated using HEK239T cells.

[0317] The resulting LV titers were quantified using a 3-day integration assay. DNA from transduced cells was harvested 3 days post-transduction, and unintegrated DNA was removed. qPCR was then used to determine and quantify the presence / integration of the vector into host cell DNA.

[0318] As shown in Figure 6, the pGM826 and pGM830 modified vector genome plasmids resulted in an observable increase in LV integration compared to the unmodified pGM326 vector genome plasmid. The pGM830 vector genome plasmid resulted in an observable increase in LV integration compared to the unmodified pGM326 vector genome plasmid, which was 9.3 x 10 5 Compared with TU / mL, the highest LV incorporation (1.3 × 10 6 TU / mL).

[0319] Again, a comparison of vector titers using either pGM326 and the modified vector genome plasmid in an otherwise identical production protocol demonstrated that use of the modified vector genome plasmid afforded at least equivalent LV integration as pGM326, indicating that an improved safety profile could be achieved without adversely affecting LV functionality. [Example]

[0320] Modifications of the vector genome plasmid, including reducing the number of intact SIV ORFs within the vector genome plasmid, maintain or even increase transgene expression. SIV-CFTR generated using pGM326 or pGM830 was used to transduce A549 cells in the presence and absence of AZT and raltegravir. All cells were stained for CFTR expression 3 days after transduction. Only cells transduced in the absence of inhibitor were then passaged and stained again for CFTR expression 10 days after transduction to examine the extent of mock transduction (transduction without proviral DNA integration into the host genome), which also resulted in CFTR expression.

[0321] As shown in Figure 7, when an inhibitor of reverse transcription (azidothymidine, AZT) and an inhibitor of SIV integration (raltegravir) were used, the number of cells expressing CFTR was almost the same as in the negative control, implying that CFTR expression was the result of LV integration.

[0322] Furthermore, Figure 7 also demonstrates that the % of CFTR-positive cells was higher for LVs generated using pGM830 compared to LVs generated using pGM326, even when AZT was included during transduction.

[0323] Therefore, this comparison of CFTR transgene expression using either pGM326 and pGM830 demonstrated that the use of the modified vector genome plasmids confers at least equivalent transgene expression compared to LVs produced using unmodified pGM326, and indicated that an improved safety profile could be achieved without adversely affecting LV functionality. [Example]

[0324] Fct4 is cleaved by an enzyme with trypsin-like cleavage specificity to generate a fusion-active form containing F1 and F2 fragments LVs produced according to Example 1 were evaluated for F protein cleavage after the addition of a trypsin-like enzyme. Activation of the F protein occurs by cleavage into two subunits, F1 and F2. Therefore, F protein cleavage is a recognized proxy for F protein activation and, therefore, fusion competence.

[0325] After incubation of LV with a trypsin-like enzyme, Western blots were performed using anti-PIV1 antibody ab20791 at a dilution of 1:5000. As shown in Figure 8, incubation with a trypsin-like enzyme successfully cleaves Fct4, ​​as uncleaved F0 was not detected in the presence of the enzyme, but rather only F1 was detected.

[0326] Sequence information Legend for Arrays SEQ ID NO: 1 Modified SIV / CFTR RNA sequence SEQ ID NO: 2 p17 protein sequence SEQ ID NO: 3 p24 protein sequence SEQ ID NO: 4 p8 protein sequence SEQ ID NO: 5 Protease sequence SEQ ID NO: 6 p51 protein sequence SEQ ID NO: 7 p15 protein sequence SEQ ID NO: 8 p31 protein sequence SEQ ID NO: 9 Gag protein SEQ ID NO: 10 Pol protein Sequence number 11 (skip) SEQ ID NO: 12 Fct4 protein SEQ ID NO: 13 Fct4 protein (including signal sequence) SEQ ID NO: 14 Fct4 protein (fragment 1) SEQ ID NO: 15 Fct4 protein (fragment 2) SEQ ID NO: 16 Fct4 protein signal sequence SEQ ID NO: 17 Codon-optimized SIV gag-pol nucleic acid sequence SEQ ID NO: 18 Wild-type SIV gag-pol nucleic acid sequence SEQ ID NO: 19 Plasmid defined in Figure 2A (pDNA1 pGM830) SEQ ID NO: 20 Plasmid defined in Figure 2B (pDNA1 pGM691) SEQ ID NO: 21 Plasmid defined in Figure 2C (pDNA2a pGM297) SEQ ID NO: 22 Plasmid defined in Figure 2D (pDNA2b pGM299) SEQ ID NO: 23 Plasmid defined in Figure 2E (pDNA3a pGM301) SEQ ID NO: 24 Plasmid defined in Figure 2F (pDNA3b pGM303) SEQ ID NO: 25 Plasmid defined in Figure 2G (pDNA2a pGM326) SEQ ID NO: 26 Exemplary hCEF promoter SEQ ID NO: 27 Exemplary CMV promoter SEQ ID NO: 28 Exemplary EF1a promoter SEQ ID NO: 29 Exemplary CFTR transgene (soCFTR2) SEQ ID NO: 30 Exemplary A1AT transgene SEQ ID NO: 31 Complement to the exemplified A1AT transgene SEQ ID NO: 32 Exemplary A1A1 Polypeptide SEQ ID NO: 33 Exemplary FVIII transgene (N6) SEQ ID NO: 34 Exemplary FVIII transgene (V3) SEQ ID NO: 35 Complement to the exemplified FVIII transgene (N6) SEQ ID NO: 36 Complement to an exemplary FVIII transgene (V3) SEQ ID NO: 37 Exemplary FVIII Polypeptide (N6) SEQ ID NO: 38 Exemplary FVIII Polypeptide (V3) SEQ ID NO: 39 Exemplary WPRE component (mWPRE) SEQ ID NO: 40 F / HN-SIV-hCEF-soA1AT plasmid (pDNA1 pGM407) as defined in Figure 3 SEQ ID NO: 41 F / HN-SIV-CMV-HFVIII-V3 plasmid (pDNA1 pGM411) as defined in Figure 4A SEQ ID NO: 42 F / HN-SIV-hCEF-HFVIII-V3 plasmid (pDNA1 pGM413) as defined in Figure 4B SEQ ID NO: 43 F / HN-SIV-CMV-HFVIII-N6-co plasmid (pDNA1 pGM412) as defined in Figure 4C SEQ ID NO: 44 F / HN-SIV-hCEF-HFVIII-N6-co plasmid (pDNA1 pGM414) as defined in Figure 4D SEQ ID NO: 45 Exemplary CAG promoter SEQ ID NO: 46 Additional amino acid sequence encoded from a pseudo transcription start site upstream of that encoding Fct4 in SEQ ID NO: 13 [Sequence table]

[0327] <210> SEQ ID NO: 1 <211> 7553 <223> Modified SIV / CFTR RNA sequence ucucuuacua ggagaccagc uugagccugg guguucgcug guuagccuaa ccuguuggc 60 caccaggggu aaggacuccu uggcuuagaa agcuaauaaa cuugccugca uuagagcuua 120 ucugaguca guguccucau ugacgccuca cucucuugaa cgggaaucuu ccuuacugggg 180 uucucucucu gaccaggcg agagaacuc cagcaguggc gcccgaacag ggacuugagu 240 gagaguguag gcacguacag cugagaaggc gucggacgcg aaggaagcgc ggggugcgac 300 gcgaccaag aggacuug gugaguaggc ucucgagug ccgggaaaaa gcucgagccu 360 aguagagga cuaggagagg ccguagccgu aacuacug ggcaaguagg gcaggcggug 420 gguacgcaau ugggggcggc uaccucagca cuaauagga ggaauuaga ccaauuugag 480 aaaauacgac uucgcccgaa cggaaagaa aaguaccaaa uaaaacauuu auauauugggc 540 agggaggg auggagcgc ucggcucc augagagguu guuggagaca gaggagggg 600 guaaaagaau cauagaaguc cucuaccccc uagaccaac aggaucggag ggcuuaaaaaa 660 gucuguuca ucugugugc gugcuauauu gcuugcacaca ggacagaaa gugaagaca 720 cagaggaagc aguagcaca guagacaac acugccaucu aguggaaaaaaaaaagug 780 siacagagac aucuagugga haaaaaaa augacaaggg aauagcagcg ccaccuggg 840 cgagucagaa uuuuccagcg caacaacaag gaauugccu ggguacaugu acccuuguca 900 ccgcgcaccu uaaaugcgug gguaaaagca guagaggaga aaaaauuugg agcagaaaua 960 1020 uggggggcugc uggaacugca uugggagcag cggcgacagc ccugacgguc cagucucagc 1080 auuugcuugc uggguaacug cagcagcaga agaaucugcu ggcggcugug gaggcucaac 1140 agcagauguu gaagcugacc auuuggggug uuaaaaaccu caaugcccgc gucacagccc 1200 uugagaaagua ccuagaggau caggcacgac uaaacuccug ggggugcgca uggaaacaag 1260 uaugucauac cacaguggag uggcccugga caaaucggac uccggauugg caaaauaaga 1320 cuugguugga gugggaaaga caaauagcug auuuggaag caacauuacg agacaauuag 1380 ugaaggcuag agaacaagag gaaaagaauc uagaugccua ucagaaguua acuaguuggu 1440 cagauuuucug gucuugguuc gauuuucucaa aauggcuuaa cauuuuaaaa aagggauuuu 1500 1560 ggguuaggca gggauauguu ccucuaucuc cacagaucca uauaaagcgg caauuuuaaa 1620 agaaagggag gaauaggggg acagacuuca gcagagagac uaauuaauau aauaacaaca 1680 caauuagaaa uacaacauuu acaaaccaaa auucaaaaaa uuuuaaauuu uagagccgcg 1740 gagaucuguu acauaacuua ugguaaaugg ccugccuggc ugacugccca augaccccug 1800 cccaaugaug ucaauaauga uguauguucc cauguaaugc caauagggac uuuccauuga 1860 ugucaauggg uggaguauuu augguaacug cccacuuggc aguacaucaa guguaucaua 1920 ugccaaguau gcccccuauu gaugucaaug augguaaaug gccugccugg cauuaugccc 1980 aguacaugac cuuaugggac uuuccuacuu ggcaguacau cuauguauua gucauugcua 2040 uuaccauggg aauucacuag uggagaagag caugcuugag ggcugagugc cccucagugg 2100 gcagagagca cauggcccac agucccugag aaguuggggg gagggguggg caauugaacu 2160 ggugccuaga gaaggugggg cuuggguaaa cugggaaagu gauguggugu acuggcucca 2220 ccuuuuuccc cagggugggg gagaaccaua uauaagugca guagucucug ugaacauuca 2280 agcuucugcc uucuccucc ugugaguuug quagccacca ugcagagag cccucuggag 2340 aaggccucug gaggagça gcugucuuc agcuggacca ggcccug gaggaagggc 2400 uacaggcaga gacuggagcu gucugacauc uaccagaucc ccucugugga cucugcugac 2460 aaccugucug agaagcugga gagggagugg gauagagagc uggccagca gagaacccc 2520 augcugauca augcccugag gagaugcuuc augcuggau augcuucuc augcuucuc 2580 cuguaccugg gggaagugac caggcugug cagcucugc ugcugggcag aucauugcc 2640 agcuaugacc cugashaacha ggaggagagg agcauugcca cugaccuggg cauggccug 2700 ugccugcugu ucauugugag gacccugcug cugcacccug ccaucuuugg cugcaccac 2760 auuggcaugc agaagaggau ugccauguuc agccugaucu ahaaaaac ccugaagcug 2820 shake shake shake gaucashauu ggshagcugg shake shake shake shake 2880 cugacaagu uugaugagggg cuggcccug gccacuuug uguggauugc cccucugcag 2940 guggcccugc ugaugggccu gauuugggag cugcugcagg cucugccuu uugggccug 3000 ggcuuccuga uugugcuggc ccuguuucag gcuggccugg gcaggaugau gaugaaguac 3060 agggaccaga gggcaggca gaucagugag agggaccaga ucaccucuga gaugauugag 3120 aacauccagu cugugaggc cucuguugg gaggaagcua uggagau gauugaaaac 3180 cugaggcaga cagagcugaa gcugaccagg aaggcugccu augugagaua cucacagc 3240 quagcuucu cucucucugg cucuucug cugugcugcc cucugcugcc cucugcug 3300 aucaagggga ucauccugag aagauuuuc accaccauca gcuucugcau ugugcugagg 3360 auggcuguga ccagacagou ccccuggggcu gugcagaccu gguaugacag ccuggggggcc 3420 aucacaaga uccaggacuu ccugcagag caggaguaca agacccugga guacaccug 3480 accaccacag aaggugau ggagaaugug accacccuucu gggagggggg 3540 cuguuugaga aggccaagca aaxagaac aaagaagg ggaugaugg ggaugacucc 3600 cuguucuuc cuckoocuc cugcugggc acccugugc ugagacau cuckoo 3660 auugagagggg ggcagcugcu ggcuguggcu ggaucuacag gggcuggca gaccagccug 3720 3780 aucagcuuuu gcagccaguu cagcuggauc augccuggca ccaucaaagga gaacaucauc 3840 3900 gagacauca ccaaguuuugc ugagaaggac aacauugugc uggggggaggg aggcauuaca 3960 cugucugggg gccagagagc cagaaucagc cuggccaggg cuguguacaa ggaugcugac 4020 cuguaccugc uggacucccc cuuuggcuac cuggaugugc ugacagagaa ggagauuuu 4080 gagagcugug ugugcaagcu gauggccaac aagaccagaa uccuggugac cagcaagaug 4140 4200 4260 gacagcuuug accaguucuc ugcugagagg aggaacagca uccugacaga gaccugcac 4320 agauucagcc uggagggaga ugccccugug agcuggacag agaccaagaa cgagagcuuc 4380 aagcagacag gggaguuugg ggagaagagg aagacucca uccugaaccc caucaacagc 4440 aucaggaagu ucagcauugu gcagaaaacc ccccugcaga ugaauggcau ugagaagagau 4500 ucugaugagc cccuggagag gagacugagc cuggugccug auucugagca gggagaggcc 4560 auccugccua ggaucucugu gaucagcaca ggcccuacac ugcaggccag aaagggcag 4620 ucugugcuga accugaugac ccacucugug aaccagggcc agaacaucca caggaaaacc 4680 acagccucca ccaggaaagu gagccuggcc ccucaggcca aucugacaga ggugcauc 4740 uacagcagga ggcugucuca ggagacaggc cuggagaauuu cugaggagau caaugaggag 4800 gaccugaaag agugcuucuu ugaugacaug gagagcaucc cugcugugac caccuggaac 4860 4920 gugaucuucc uggcugaagu ggcugcccu cugguggugc uguggcugcu gggaaacacc 4980 ccacugcagg acaagggcaa cagcacccac agcaggaaca acagcuaugc ugugaucauc 5040 accuccaccu ccagcuacua ugugucuac aucuaugugg gaguggcuga uacccugcug 5100 gcuaugggcu ucuuuagagg ccugccccug gugcacac ugaucacagu gagcaagauc 5160 cuccaccaca agaugcugca cucugugcug caggcuccua ugagcacccu gaauacccug 5220 aaggcugggg gcauccugaa cagauucucc aagcauauug ccauccugga ugaccugcug 5280 ccucucacca ucuuugacuu cauccagcug cugcugauug ugauuggggc cauugcugug 5340 guggcagugc ugcagcccua caucuuugug gccacagugc cugugauugu ggccuucauc 5400 augcugaggg ccuacuuucu gcagaccucc cagcagcuga agcagcugga gucugagggc 5460 agaagcccca ucuucaccca ccuggugaca agccugaagg gccuguggac ccugagagcc 5520 uuuggcaggc agcccuacuu ugacaccug uuccacaagg cccugaaccu gcacacagcc 5580 aacugguucc ucuaccuguc cacccugaga ugguuccaga ugagaauuga gaugaucuuu 5640 gucaucuucu ucauugcugu gaccuucauc agcauucuga ccacaggaga gggagagggc 5700 agagugggca uuauccugac ccuggccaug aacaucauga gcacacugca gugggcagug 5760 aacagcagca uugaugugga cagccugaug aggaguguga gcagaguguu caaguucauu 5820 gauaugccca cagagggcaa gccuaccaag agcaccaagc ccuacaagaa uggccagcug 5880 agcaaaguga ugaucauuga gaacagccau gugaagaagg augauaucug gcccagugga 5940 ggccagauga cagugaagga ccugacagcc aaguacacag aggggggcaa ugcuauccug 6000 gagaacaucu ccuucagcau cuccccuggc cagagagugg gacugcuggg aagaacaggc 6060 ucuggcaagu cuacccugcu gucugccuuc cugaggcugc ugaacacaga gggagagauc 6120 cagauugaug gaguguccug ggacagcauc acacugcagc agguggagaa ggccuuuggu 6180 gugauccccc agaaaguguu caucuucagu ggcaccuuca ggaagaaccu ggaccccuau 6240 gagcaguggu cugaccagga gauuuggaaa guggcugaug aagugggccu gagaagugug 6300 auugagcagu ucccuggcaa gcuggacuuu guccuggugg auggggcug uggcugagc 6360 cauggccaca agcagcugau gugccuggcc agaucagugc ugagcaaggc caagauccug 6420 cugcuggaug agccuucugc ccaccuggau ccugugaccu accagaucau caggaggacc 6480 cucaagcagg ccuuugcuga cugcacaguc auccugug agcacaggau ugaggccaug 6540 cuggagugcc agcaguuccu ggugauugag gagaacaaag ugaggcagua ugacagcauc 6600 6660 aagcuguucc cccacaggaa cagcuccaag ugcaagagca agccccagau ugcugcccug 6720 aaggagga cagaggagga agugcaggac accaggcugu gagggcccaa ucaaccucug 6780 gauuacaaaa uuugugaaag auugacuggu auucuuacu auguugcucc uuuuacgcua 6840 uggguaacg cugcuuuau gccuuuguau caugcuauug cuucccguau ggcuuuucauu 6900 uucuccuccu uguauaaauc cugguugcug ucucuuuaug aggaguugug gcccguuguc 6960 aggcaacgug gcguggugug cacuguguuu gcugacgcaa cccccacugg uuggggcauu 7020 gccaccaccu gucagcuccu uuccgggacu uucgcuuucc cccuccuau ugccacggcg 7080 gaacucaucg ccgccugccu ugcccgcugc uggacagggg cucggcuguu gggcacugac 7140 aauuccgugg uguugucggg gaaucaucg uccuuuccuu ggcugcucgc cuguuguugcc 7200 accuggauuc ugcgcgggac guccuucugc uacgucccuu cggcccuacaa uccagcggac 7260 cuuccuuccc gcggccugcu gccggcucug cggccucuuc cgcgucuucg ccuucgcccu 7320 cagacgaguc ggaucucccu uugggccgcc uccccgcaag cucgcacuu uuuaaaagaaa 7380 aagggaggac uggaugggau uuauuacucc gauaggacgc uggcuuguaa cucagucucucu 7440 uacuaggaga ccugcuugag ccuggguguu cgcugguuag ccuaccugg uuggccacca 7500 gggguaagga cucuuggcu uagaaagcua auaaacuugc cugcauuaga gcu 7553 <210> SEQ ID NO: 2 <211> 140 <223> p17 protein Gly Ala Ala Thr Ser Ala Leu Asn Arg Arg Gln Leu Asp Gln Phe Glu 1 5 10 15 Lys Ile Arg Leu Arg Pro Asn Gly Lys Lys Tyr Gln Ile Lys His 20 25 30 Leu Ile Trp Ala Gly Lys Glu Met Glu Arg Phe Gly Leu His Glu Arg 35 40 45 Leu Glu Thr Glu Glu Gly Cys Lys Arg Ile Ile Glu Val Leu Tyr 50 55 60 Pro Leu Glu Pro Thr Gly Ser Glu Gly Leu Lys Ser Leu Phe Asn Leu 65 70 75 80 Will Cys Will Tyr Cys Lew His Lys Glu Gln Lys Will Lys Asp Thr 85 90 95 Glu Glu Ala Val Ala Thr Val Arg Gln His Cys His Leu Val Glu Lys 100 105 110 Glu Lys Ser Ala Thr Glu Thr Ser Ser Gly Gln Lys Lys Asn Asp Lys 115 120 125 Gly Ile Ala Ala Pro Pro Gly Gly Ser Gln Asn Phe 130 135 140 <210> SEQ ID NO: 3 <211> 231 <223> p24 protein Pro Ala Gln Gln Gln Gly Asn Ala Trp Val His Val Pro Leu Ser Pro 1 5 10 15 Arg Thr Leu Asn Ala Trp Val Lys Ala Val Glu Glu Lys Lys Phe Gly 20 25 30 Ala Glu Ile Val Pro Met Phe Gln Ala Leu Ser Glu Gly Cys Thr Pro 35 40 45 Tyr Asp Ile Asn Gln Met Leu Asn Val Leu Gly Asp His Gln Gly Ala 50 55 60 Leu Gln Ile Val Lys Glu Ile Ile Asn Glu Glu Ala Ala Gln Trp Asp 65 70 75 80 Val Thr His Pro Leu Pro Ala Gly Pro Leu Pro Ala Gly Gln Leu Arg 85 90 95 Asp Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Ser Val Gln Glu 100 105 110 Gln Leu Glu Trp Ile Tyr Thr Ala Asn Pro Arg Val Asp Val Gly Ala 115 120 125 Ile Tyr Arg Arg Trp Ile Ile Leu Gly Leu Gln Lys Cys Val Lys Met 130 135 140 Tyr Asn Pro Val Ser Val Leu Asp Ile Arg Gln Gly Pro Lys Glu Pro 145 150 155 160 Phe Lys Asp Tyr Val Asp Arg Phe Tyr Lys Ala Ile Arg Ala Glu Gln 165 170 175 Ala Ser Gly Glu Val Lys Gln Trp Met Thr Glu Ser Leu Leu Ile Gln 180 185 190 Asn Ala Asn Pro Asp Cys Lys Val Ile Leu Lys Gly Leu Gly Met His 195 200 205 Pro Thr Leu Glu Glu Met Leu Thr Ala Cys Gln Gly Val Gly Gly Pro 210 215 220 Ser Tyr Lys Ala Lys Val Met 225 230 <210> SEQ ID NO: 4 <211> 54 <223> p8 protein Val Gln Gln Gly Gly Pro Lys Arg Gln Arg Pro Pro Leu Arg Cys Tyr 1 5 10 15 Asn Cys Gly Lys Phe Gly His Met Gln Arg Gln Cys Pro Glu Pro Arg 20 25 30 Lys Thr Lys Cys Leu Lys Cys Gly Lys Leu Gly His Leu Ala Lys Asp 35 40 45 Cys Arg Gly Gln Val Asn 50 <210> SEQ ID NO: 5 <211> 101 <223> protease Phe Glu Leu Pro Leu Trp Arg Arg Pro Ile Lys Thr Val Tyr Ile Glu 1 5 10 15 Gly Val Pro Ile Lys Ala Leu Leu Asp Thr Gly Ala Asp Asp Thr Ile 20 25 30 Ile Lys Glu Asn Asp Leu Gln Leu Ser Gly Pro Trp Arg Pro Lys Ile 35 40 45 Ile Gly Gly Ile Gly Gly Gly Leu Asn Val Lys Glu Tyr Asn Asp Arg 50 55 60 Glu Val Lys Ile Glu Asp Lys Ile Leu Arg Gly Thr Ile Leu Leu Gly 65 70 75 80 Ala Thr Pro Ile Asn Ile Ile Gly Arg Asn Leu Leu Ala Pro Ala Gly 85 90 95 Ala Arg Leu Val Met 100 <210> SEQ ID NO: 6 <211> 441 <223> p51 protein Gly Gln Leu Ser Glu Lys Ile Pro Val Thr Pro Val Lys Leu Lys Glu 1 5 10 15 Gly Ala Arg Gly Pro Cys Val Arg Gln Trp Pro Leu Ser Lys Glu Lys 20 25 30 Ile Glu Ala Leu Gln Glu Ile Cys Ser Gln Leu Glu Gln Glu Gly Lys 35 40 45 Ile Ser Arg Val Gly Gly Glu Asn Ala Tyr Asn Thr Pro Ile Phe Cys 50 55 60 Ile Lys Lys Lys Asp Lys Ser Gln Trp Arg Met Leu Val Asp Phe Arg 65 70 75 80 Glu Leu Asn Lys Ala Thr Gln Asp Phe Phe Glu Val Gln Leu Gly Ile 85 90 95 Pro His Pro Ala Gly Leu Arg Lys Met Arg Gln Ile Thr Val Leu Asp 100 105 110 Val Gly Asp Ala Tyr Tyr Ser Ile Pro Leu Asp Pro Asn Phe Arg Lys 115 120 125 Tyr Thr Ala Phe Thr Ile Pro Thr Val Asn Asn Gln Gly Pro Gly Ile 130 135 140 Arg Tyr Gln Phe Asn Cys Leu Pro Gln Gly Trp Lys Gly Ser Pro Thr 145 150 155 160 Ile Phe Gln Asn Thr Ala Ala Ser Ile Leu Glu Glu Ile Lys Arg Asn 165 170 175 Leu Pro Ala Leu Thr Ile Val Gln Tyr Met Asp Asp Leu Trp Val Gly 180 185 190 Ser Gln Glu Asn Glu His Thr His Asp Lys Leu Val Glu Gln Leu Arg 195 200 205 Thr Lys Leu Gln Ala Trp Gly Leu Glu Thr Pro Glu Lys Lys Val Gln 210 215 220 Lys Glu Pro Pro Tyr Glu Trp Met Gly Tyr Lys Leu Trp Pro His Lys 225 230 235 240 Trp Glu Leu Ser Arg Ile Gln Leu Glu Glu Lys Asp Glu Trp Thr Val 245 250 255 Asn Asp Ile Gln Lys Leu Val Gly Lys Leu Asn Trp Ala Ala Gln Leu 260 265 270 Tyr Pro Gly Leu Arg Thr Lys Asn Ile Cys Lys Leu Ile Arg Gly Lys 275 280 285 Lys Asn Leu Leu Glu Leu Val Thr Trp Thr Pro Glu Ala Glu Ala Glu 290 295 300 Tyr Ala Glu Asn Ala Glu Ile Leu Lys Thr Glu Gln Glu Gly Thr Tyr 305 310 315 320 Tyr Lys Pro Gly Ile Pro Ile Arg Ala Ala Val Gln Lys Leu Glu Gly 325 330 335 Gly Gln Trp Ser Tyr Gln Phe Lys Gln Glu Gly Gln Val Leu Lys Val 340 345 350 Gly Lys Tyr Thr Lys Gln Lys Asn Thr His Thr Asn Glu Leu Arg Thr 355 360 365 Leu Ala Gly Leu Val Gln Lys Ile Cys Lys Glu Ala Leu Val Ile Trp 370 375 380 Gly Ile Leu Pro Val Leu Glu Leu Pro Ile Glu Arg Glu Val Trp Glu 385 390 395 400 Gln Trp Trp Ala Asp Tyr Trp Gln Val Ser Trp Ile Pro Glu Trp Asp 405 410 415 Phe Val Ser Thr Pro Pro Leu Leu Lys Leu Trp Tyr Thr Leu Thr Lys 420 425 430 Glu Pro Ile Pro Lys Glu Asp Val Tyr 435 440 <210> SEQ ID NO: 7 <211> 120 <223> p15 protein Tyr Val Asp Gly Ala Cys Asn Arg Asn Ser Lys Glu Gly Lys Ala Gly 1 5 10 15 Tyr Ile Ser Gln Tyr Gly Lys Gln Arg Val Glu Thr Leu Glu Asn Thr 20 25 30 Thr Asn Gln Gln Ala Glu Leu Thr Ala Ile Lys Met Ala Leu Glu Asp 35 40 45 Ser Gly Pro Asn Val Asn Ile Val Thr Asp Ser Gln Tyr Ala Met Gly 50 55 60 Ile Leu Thr Ala Gln Pro Thr Gln Ser Asp Ser Pro Leu Val Glu Gln 65 70 75 80 Ile Ile Ala Leu Met Ile Gln Lys Gln Gln Ile Tyr Leu Gln Trp Val 85 90 95 Pro Ala His Lys Gly Ile Gly Gly Asn Glu Glu Ile Asp Lys Leu Val 100 105 110 Ser Lys Gly Ile Arg Arg Val Leu 115 120 <210> SEQ ID NO: 8 <211> 291 <223> p31 protein Phe Leu Glu Lys Ile Glu Glu Ala Gln Glu Glu His Glu Arg Tyr His 1 5 10 15 Asn Asn Trp Lys Asn Leu Ala Asp Thr Tyr Gly Leu Pro Gln Ile Val 20 25 30 Ala Lys Glu Ile Val Ala Met Cys Pro Lys Cys Gln Ile Lys Gly Glu 35 40 45 Pro Val His Gly Gln Val Asp Ala Ser Pro Gly Thr Trp Gln Met Asp 50 55 60 Cys Thr His Leu Glu Gly Lys Val Val Ile Val Ala Val His Val Ala 65 70 75 80 Ser Gly Phe Ile Glu Ala Glu Val Ile Pro Arg Glu Thr Gly Lys Glu 85 90 95 Thr Ala Lys Phe Leu Leu Lys Ile Leu Ser Arg Trp Pro Ile Thr Gln 100 105 110 Leu His Thr Asp Asn Gly Pro Asn Phe Thr Ser Gln Glu Val Ala Ala 115 120 125 Ile Cys Trp Trp Gly Lys Ile Glu His Thr Thr Gly Ile Pro Tyr Asn 130 135 140 Pro Gln Ser Gln Gly Ser Ile Glu Ser Met Asn Lys Gln Leu Lys Glu 145 150 155 160 Ile Ile Gly Lys Ile Arg Asp Asp Cys Gln Tyr Thr Glu Thr Ala Val 165 170 175 Leu Met Ala Cys His Ile His Asn Phe Lys Arg Lys Gly Gly Ile Gly 180 185 190 Gly Gln Thr Ser Ala Glu Arg Leu Ile Asn Ile Ile Thr Thr Gln Leu 195 200 205 Glu Ile Gln His Leu Gln Thr Lys Ile Gln Lys Ile Leu Asn Phe Arg 210 215 220 Val Tyr Tyr Arg Glu Gly Arg Asp Pro Val Trp Lys Gly Pro Ala Gln 225 230 235 240 Leu Ile Trp Lys Gly Glu Gly Ala Val Val Leu Lys Asp Gly Ser Asp 245 250 255 Leu Lys Val Val Pro Arg Arg Lys Ala Lys Ile Ile Lys Asp Tyr Glu 260 265 270 Pro Lys Gln Arg Val Gly Asn Glu Gly Asp Val Glu Gly Thr Arg Gly 275 280 285 Ser Asp Asn 290 <210> SEQ ID NO: 9 <211> 519 <223> Gag protein Met Gly Ala Ala Thr Ser Ala Leu Asn Arg Arg Gln Leu Asp Gln Phe 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Asn Gly Lys Lys Lys Tyr Gln Ile Lys 20 25 30 His Leu Ile Trp Ala Gly Lys Glu Met Glu Arg Phe Gly Leu His Glu 35 40 45 Arg Leu Leu Glu Thr Glu Glu Gly Cys Lys Arg Ile Ile Glu Val Leu 50 55 60 Tyr Pro Leu Glu Pro Thr Gly Ser Glu Gly Leu Lys Ser Leu Phe Asn 65 70 75 80 Leu Val Cys Val Leu Tyr Cys Leu His Lys Glu Gln Lys Val Lys Asp 85 90 95 Thr Glu Glu Ala Val Ala Thr Val Arg Gln His Cys His Leu Val Glu 100 105 110 Lys Glu Lys Ser Ala Thr Glu Thr Ser Ser Gly Gln Lys Lys Asn Asp 115 120 125 Lys Gly Ile Ala Ala Pro Pro Gly Gly Ser Gln Asn Phe Pro Ala Gln 130 135 140 Gln Gln Gly Asn Ala Trp Val His Val Pro Leu Ser Pro Arg Thr Leu 145 150 155 160 Asn Ala Trp Val Lys Ala Val Glu Glu Lys Lys Phe Gly Ala Glu Ile 165 170 175 Val Pro Met Phe Gln Ala Leu Ser Glu Gly Cys Thr Pro Tyr Asp Ile 180 185 190 Asn Gln Met Leu Asn Val Leu Gly Asp His Gln Gly Ala Leu Gln Ile 195 200 205 Val Lys Glu Ile Ile Asn Glu Glu Ala Ala Gln Trp Asp Val Thr His 210 215 220 Pro Leu Pro Ala Gly Pro Leu Pro Ala Gly Gln Leu Arg Asp Pro Arg 225 230 235 240 Gly Ser Asp Ile Ala Gly Thr Thr Ser Ser Val Gln Glu Gln Leu Glu 245 250 255 Trp Ile Tyr Thr Ala Asn Pro Arg Val Asp Val Gly Ala Ile Tyr Arg 260 265 270 Arg Trp Ile Ile Leu Gly Leu Gln Lys Cys Val Lys Met Tyr Asn Pro 275 280 285 Val Ser Val Leu Asp Ile Arg Gln Gly Pro Lys Glu Pro Phe Lys Asp 290 295 300 Tyr Val Asp Arg Phe Tyr Lys Ala Ile Arg Ala Glu Gln Ala Ser Gly 305 310 315 320 Glu Val Lys Gln Trp Met Thr Glu Ser Leu Leu Ile Gln Asn Ala Asn 325 330 335 Pro Asp Cys Lys Val Ile Leu Lys Gly Leu Gly Met His Pro Thr Leu 340 345 350 Glu Glu Met Leu Thr Ala Cys Gln Gly Val Gly Gly Pro Ser Tyr Lys 355 360 365 Ala Lys Val Met Ala Glu Met Met Gln Thr Met Gln Asn Gln Asn Met 370 375 380 Val Gln Gln Gly Gly Pro Lys Arg Gln Arg Pro Pro Leu Arg Cys Tyr 385 390 395 400 Asn Cys Gly Lys Phe Gly His Met Gln Arg Gln Cys Pro Glu Pro Arg 405 410 415 Lys Thr Lys Cys Leu Lys Cys Gly Lys Leu Gly His Leu Ala Lys Asp 420 425 430 Cys Arg Gly Gln Val Asn Phe Leu Gly Tyr Gly Arg Trp Met Gly Ala 435 440 445 Lys Pro Arg Asn Phe Pro Ala Ala Thr Leu Gly Ala Glu Pro Ser Ala 450 455 460 Pro Pro Pro Pro Ser Gly Thr Thr Pro Tyr Asp Pro Ala Lys Lys Leu 465 470 475 480 Leu Gln Gln Tyr Ala Glu Lys Gly Lys Gln Leu Arg Glu Gln Lys Arg 485 490 495 Asn Pro Pro Ala Met Asn Pro Asp Trp Thr Glu Gly Tyr Ser Leu Asn 500 505 510 Ser Leu Phe Gly Glu Asp Gln 515 <210> SEQ ID NO: 10 <211> 1044 <223> Pol protein Met Ser Lys Val Trp Lys Ile Gly Thr Pro Ser Lys Arg Leu Gln Gly 1 5 10 15 Thr Gly Glu Phe Phe Arg Val Trp Thr Val Asp Gly Gly Lys Thr Glu 20 25 30 Lys Phe Ser Arg Arg Tyr Ser Trp Ser Gly Thr Glu Cys Ala Ser Ser 35 40 45 Thr Glu Arg His His Pro Ile Arg Pro Ser Lys Glu Ala Pro Ala Ala 50 55 60 Ile Cys Arg Glu Arg Glu Thr Thr Glu Gly Ala Lys Glu Glu Ser Thr 65 70 75 80 Gly Asn Glu Ser Gly Leu Asp Arg Gly Ile Phe Phe Glu Leu Pro Leu 85 90 95 Trp Arg Arg Pro Ile Lys Thr Val Tyr Ile Glu Gly Val Pro Ile Lys 100 105 110 Ala Leu Leu Asp Thr Gly Ala Asp Asp Thr Ile Ile Lys Glu Asn Asp 115 120 125 Leu Gln Leu Ser Gly Pro Trp Arg Pro Lys Ile Ile Gly Gly Ile Gly 130 135 140 Gly Gly Leu Asn Val Lys Glu Tyr Asn Asp Arg Glu Val Lys Ile Glu 145 150 155 160 Asp Lys Ile Leu Arg Gly Thr Ile Leu Leu Gly Ala Thr Pro Ile Asn 165 170 175 Ile Ile Gly Arg Asn Leu Leu Ala Pro Ala Gly Ala Arg Leu Val Met 180 185 190 Gly Gln Leu Ser Glu Lys Ile Pro Val Thr Pro Val Lys Leu Lys Glu 195 200 205 Gly Ala Arg Gly Pro Cys Val Arg Gln Trp Pro Leu Ser Lys Glu Lys 210 215 220 Ile Glu Ala Leu Gln Glu Ile Cys Ser Gln Leu Glu Gln Glu Gly Lys 225 230 235 240 Ile Ser Arg Val Gly Gly Glu Asn Ala Tyr Asn Thr Pro Ile Phe Cys 245 250 255 Ile Lys Lys Lys Asp Lys Ser Gln Trp Arg Met Leu Val Asp Phe Arg 260 265 270 Glu Leu Asn Lys Ala Thr Gln Asp Phe Phe Glu Val Gln Leu Gly Ile 275 280 285 Pro His Pro Ala Gly Leu Arg Lys Met Arg Gln Ile Thr Val Leu Asp 290 295 300 Val Gly Asp Ala Tyr Tyr Ser Ile Pro Leu Asp Pro Asn Phe Arg Lys 305 310 315 320 Tyr Thr Ala Phe Thr Ile Pro Thr Val Asn Asn Gln Gly Pro Gly Ile 325 330 335 Arg Tyr Gln Phe Asn Cys Leu Pro Gln Gly Trp Lys Gly Ser Pro Thr 340 345 350 Ile Phe Gln Asn Thr Ala Ala Ser Ile Leu Glu Glu Ile Lys Arg Asn 355 360 365 Leu Pro Ala Leu Thr Ile Val Gln Tyr Met Asp Asp Leu Trp Val Gly 370 375 380 Ser Gln Glu Asn Glu His Thr His Asp Lys Leu Val Glu Gln Leu Arg 385 390 395 400 Thr Lys Leu Gln Ala Trp Gly Leu Glu Thr Pro Glu Lys Lys Val Gln 405 410 415 Lys Glu Pro Pro Tyr Glu Trp Met Gly Tyr Lys Leu Trp Pro His Lys 420 425 430 Trp Glu Leu Ser Arg Ile Gln Leu Glu Glu Lys Asp Glu Trp Thr Val 435 440 445 Asn Asp Ile Gln Lys Leu Val Gly Lys Leu Asn Trp Ala Ala Gln Leu 450 455 460 Tyr Pro Gly Leu Arg Thr Lys Asn Ile Cys Lys Leu Ile Arg Gly Lys 465 470 475 480 Lys Asn Leu Leu Glu Leu Val Thr Trp Thr Pro Glu Ala Glu Ala Glu 485 490 495 Tyr Ala Glu Asn Ala Glu Ile Leu Lys Thr Glu Gln Glu Gly Thr Tyr 500 505 510 Tyr Lys Pro Gly Ile Pro Ile Arg Ala Ala Val Gln Lys Leu Glu Gly 515 520 525 Gly Gln Trp Ser Tyr Gln Phe Lys Gln Glu Gly Gln Val Leu Lys Val 530 535 540 Gly Lys Tyr Thr Lys Gln Lys Asn Thr His Thr Asn Glu Leu Arg Thr 545 550 555 560 Leu Ala Gly Leu Val Gln Lys Ile Cys Lys Glu Ala Leu Val Ile Trp 565 570 575 Gly Ile Leu Pro Val Leu Glu Leu Pro Ile Glu Arg Glu Val Trp Glu 580 585 590 Gln Trp Trp Ala Asp Tyr Trp Gln Val Ser Trp Ile Pro Glu Trp Asp 595 600 605 Phe Val Ser Thr Pro Pro Leu Leu Lys Leu Trp Tyr Thr Leu Thr Lys 610 615 620 Glu Pro Ile Pro Lys Glu Asp Val Tyr Tyr Val Asp Gly Ala Cys Asn 625 630 635 640 Arg Asn Ser Lys Glu Gly Lys Ala Gly Tyr Ile Ser Gln Tyr Gly Lys 645 650 655 Gln Arg Val Glu Thr Leu Glu Asn Thr Thr Asn Gln Gln Ala Glu Leu 660 665 670 Thr Ala Ile Lys Met Ala Leu Glu Asp Ser Gly Pro Asn Val Asn Ile 675 680 685 Val Thr Asp Ser Gln Tyr Ala Met Gly Ile Leu Thr Ala Gln Pro Thr 690 695 700 Gln Ser Asp Ser Pro Leu Val Glu Gln Ile Ile Ala Leu Met Ile Gln 705 710 715 720 Lys Gln Gln Ile Tyr Leu Gln Trp Val Pro Ala His Lys Gly Ile Gly 725 730 735 Gly Asn Glu Glu Ile Asp Lys Leu Val Ser Lys Gly Ile Arg Arg Val 740 745 750 Leu Phe Leu Glu Lys Ile Glu Glu Ala Gln Glu Glu His Glu Arg Tyr 755 760 765 His Asn Asn Trp Lys Asn Leu Ala Asp Thr Tyr Gly Leu Pro Gln Ile 770 775 780 Val Ala Lys Glu Ile Val Ala Met Cys Pro Lys Cys Gln Ile Lys Gly 785 790 795 800 Glu Pro Val His Gly Gln Val Asp Ala Ser Pro Gly Thr Trp Gln Met 805 810 815 Asp Cys Thr His Leu Glu Gly Lys Val Val Ile Val Ala Val His Val 820 825 830 Ala Ser Gly Phe Ile Glu Ala Glu Val Ile Pro Arg Glu Thr Gly Lys 835 840 845 Glu Thr Ala Lys Phe Leu Leu Lys Ile Leu Ser Arg Trp Pro Ile Thr 850 855 860 Gln Leu His Thr Asp Asn Gly Pro Asn Phe Thr Ser Gln Glu Val Ala 865 870 875 880 Ala Ile Cys Trp Trp Gly Lys Ile Glu His Thr Thr Gly Ile Pro Tyr 885 890 895 Asn Pro Gln Ser Gln Gly Ser Ile Glu Ser Met Asn Lys Gln Leu Lys 900 905 910 Glu Ile Ile Gly Lys Ile Arg Asp Asp Cys Gln Tyr Thr Glu Thr Ala 915 920 925 Val Leu Met Ala Cys His Ile His Asn Phe Lys Arg Lys Gly Gly Ile 930 935 940 Gly Gly Gln Thr Ser Ala Glu Arg Leu Ile Asn Ile Ile Thr Thr Gln 945 950 955 960 Leu Glu Ile Gln His Leu Gln Thr Lys Ile Gln Lys Ile Leu Asn Phe 965 970 975 Arg Val Tyr Tyr Arg Glu Gly Arg Asp Pro Val Trp Lys Gly Pro Ala 980 985 990 Gln Leu Ile Trp Lys Gly Glu Gly Ala Val Val Leu Lys Asp Gly Ser 995 1000 1005 Asp Leu Lys Val Val Pro Arg Arg Lys Ala Lys Ile Ile Lys Asp 1010 1015 1020 Tyr Glu Pro Lys Gln Arg Val Gly Asn Glu Gly Asp Val Glu Gly 1025 1030 1035 Thr Arg Gly Ser Asp Asn 1040 <210> SEQ ID NO: 11 <211> 0 <212> 000 <223> 000 <210> SEQ ID NO: 12 <211> 502 <223> Fct4 protein Gln Ile Pro Arg Asp Arg Leu Ser Asn Ile Gly Val Ile Val Asp Glu 1 5 10 15 Gly Lys Ser Leu Lys Ile Ala Gly Ser His Glu Ser Arg Tyr Ile Val 20 25 30 Leu Ser Leu Val Pro Gly Val Asp Phe Glu Asn Gly Cys Gly Thr Ala 35 40 45 Gln Val Ile Gln Tyr Lys Ser Leu Leu Asn Arg Leu Leu Ile Pro Leu 50 55 60 Arg Asp Ala Leu Asp Leu Gln Glu Ala Leu Ile Thr Val Thr Asn Asp 65 70 75 80 Thr Thr Gln Asn Ala Gly Ala Pro Gln Ser Arg Phe Phe Gly Ala Val 85 90 95 Ile Gly Thr Ile Ala Leu Gly Val Ala Thr Ser Ala Gln Ile Thr Ala 100 105 110 Gly Ile Ala Leu Ala Glu Ala Arg Glu Ala Lys Arg Asp Ile Ala Leu 115 120 125 Ile Lys Glu Ser Met Thr Lys Thr His Lys Ser Ile Glu Leu Leu Gln 130 135 140 Asn Ala Val Gly Glu Gln Ile Leu Ala Leu Lys Thr Leu Gln Asp Phe 145 150 155 160 Val Asn Asp Glu Ile Lys Pro Ala Ile Ser Glu Leu Gly Cys Glu Thr 165 170 175 Ala Ala Leu Arg Leu Gly Ile Lys Leu Thr Gln His Tyr Ser Glu Leu 180 185 190 Leu Thr Ala Phe Gly Ser Asn Phe Gly Thr Ile Gly Glu Lys Ser Leu 195 200 205 Thr Leu Gln Ala Leu Ser Ser Leu Tyr Ser Ala Asn Ile Thr Glu Ile 210 215 220 Met Thr Thr Ile Arg Thr Gly Gln Ser Asn Ile Tyr Asp Val Ile Tyr 225 230 235 240 Thr Glu Gln Ile Lys Gly Thr Val Ile Asp Val Asp Leu Glu Arg Tyr 245 250 255 Met Val Thr Leu Ser Val Lys Ile Pro Ile Leu Ser Glu Val Pro Gly 260 265 270 Val Leu Ile His Lys Ala Ser Ser Ile Ser Tyr Asn Ile Asp Gly Glu 275 280 285 Glu Trp Tyr Val Thr Val Pro Ser His Ile Leu Ser Arg Ala Ser Phe 290 295 300 Leu Gly Gly Ala Asp Ile Thr Asp Cys Val Glu Ser Arg Leu Thr Tyr 305 310 315 320 Ile Cys Pro Arg Asp Pro Ala Gln Leu Ile Pro Asp Ser Gln Gln Lys 325 330 335 Cys Ile Leu Gly Asp Thr Thr Arg Cys Pro Val Thr Lys Val Val Asp 340 345 350 Ser Leu Ile Pro Lys Phe Ala Phe Val Asn Gly Gly Val Val Ala Asn 355 360 365 Cys Ile Ala Ser Thr Cys Thr Cys Gly Thr Gly Arg Arg Pro Ile Ser 370 375 380 Gln Asp Arg Ser Lys Gly Val Val Phe Leu Thr His Asp Asn Cys Gly 385 390 395 400 Leu Ile Gly Val Asn Gly Val Glu Leu Tyr Ala Asn Arg Arg Gly His 405 410 415 Asp Ala Thr Trp Gly Val Gln Asn Leu Thr Val Gly Pro Ala Ile Ala 420 425 430 Ile Arg Pro Val Asp Ile Ser Leu Asn Leu Ala Asp Ala Thr Asn Phe 435 440 445 Leu Gln Asp Ser Lys Ala Glu Leu Glu Lys Ala Arg Lys Ile Leu Ser 450 455 460 Glu Val Gly Arg Trp Tyr Asn Ser Arg Glu Thr Val Ile Thr Ile Ile 465 470 475 480 Val Val Met Val Val Ile Leu Val Val Ile Ile Val Ile Ile Ile Val 485 490 495 Leu Tyr Arg Leu Arg Arg 500 <210> SEQ ID NO: 13 <211> 527 <223> Fct4 (including signal sequence) Met Ala Thr Tyr Ile Gln Arg Val Gln Cys Ile Ser Thr Ser Leu Leu 1 5 10 15 Val Val Leu Thr Thr Leu Val Ser Cys Gln Ile Pro Arg Asp Arg Leu 20 25 30 Ser Asn Ile Gly Val Ile Val Asp Glu Gly Lys Ser Leu Lys Ile Ala 35 40 45 Gly Ser His Glu Ser Arg Tyr Ile Val Leu Ser Leu Val Pro Gly Val 50 55 60 Asp Phe Glu Asn Gly Cys Gly Thr Ala Gln Val Ile Gln Tyr Lys Ser 65 70 75 80 Leu Leu Asn Arg Leu Leu Ile Pro Leu Arg Asp Ala Leu Asp Leu Gln 85 90 95 Glu Ala Leu Ile Thr Val Thr Asn Asp Thr Thr Gln Asn Ala Gly Ala 100 105 110 Pro Gln Ser Arg Phe Phe Gly Ala Val Ile Gly Thr Ile Ala Leu Gly 115 120 125 Val Ala Thr Ser Ala Gln Ile Thr Ala Gly Ile Ala Leu Ala Glu Ala 130 135 140 Arg Glu Ala Lys Arg Asp Ile Ala Leu Ile Lys Glu Ser Met Thr Lys 145 150 155 160 Thr His Lys Ser Ile Glu Leu Leu Gln Asn Ala Val Gly Glu Gln Ile 165 170 175 Leu Ala Leu Lys Thr Leu Gln Asp Phe Val Asn Asp Glu Ile Lys Pro 180 185 190 Ala Ile Ser Glu Leu Gly Cys Glu Thr Ala Ala Leu Arg Leu Gly Ile 195 200 205 Lys Leu Thr Gln His Tyr Ser Glu Leu Leu Thr Ala Phe Gly Ser Asn 210 215 220 Phe Gly Thr Ile Gly Glu Lys Ser Leu Thr Leu Gln Ala Leu Ser Ser 225 230 235 240 Leu Tyr Ser Ala Asn Ile Thr Glu Ile Met Thr Thr Ile Arg Thr Gly 245 250 255 Gln Ser Asn Ile Tyr Asp Val Ile Tyr Thr Glu Gln Ile Lys Gly Thr 260 265 270 Val Ile Asp Val Asp Leu Glu Arg Tyr Met Val Thr Leu Ser Val Lys 275 280 285 Ile Pro Ile Leu Ser Glu Val Pro Gly Val Leu Ile His Lys Ala Ser 290 295 300 Ser Ile Ser Tyr Asn Ile Asp Gly Glu Glu Trp Tyr Val Thr Val Pro 305 310 315 320 Ser His Ile Leu Ser Arg Ala Ser Phe Leu Gly Gly Ala Asp Ile Thr 325 330 335 Asp Cys Val Glu Ser Arg Leu Thr Tyr Ile Cys Pro Arg Asp Pro Ala 340 345 350 Gln Leu Ile Pro Asp Ser Gln Gln Lys Cys Ile Leu Gly Asp Thr Thr 355 360 365 Arg Cys Pro Val Thr Lys Val Val Asp Ser Leu Ile Pro Lys Phe Ala 370 375 380 Phe Val Asn Gly Gly Val Val Ala Asn Cys Ile Ala Ser Thr Cys Thr 385 390 395 400 Cys Gly Thr Gly Arg Arg Pro Ile Ser Gln Asp Arg Ser Lys Gly Val 405 410 415 Val Phe Leu Thr His Asp Asn Cys Gly Leu Ile Gly Val Asn Gly Val 420 425 430 Glu Leu Tyr Ala Asn Arg Arg Gly His Asp Ala Thr Trp Gly Val Gln 435 440 445 Asn Leu Thr Val Gly Pro Ala Ile Ala Ile Arg Pro Val Asp Ile Ser 450 455 460 Leu Asn Leu Ala Asp Ala Thr Asn Phe Leu Gln Asp Ser Lys Ala Glu 465 470 475 480 Leu Glu Lys Ala Arg Lys Ile Leu Ser Glu Val Gly Arg Trp Tyr Asn 485 490 495 Ser Arg Glu Thr Val Ile Thr Ile Ile Val Val Met Val Val Ile Leu 500 505 510 Val Val Ile Ile Val Ile Ile Ile Val Leu Tyr Arg Leu Arg Arg 515 520 525 <210> SEQ ID NO: 14 <211> 411 <223> Fct4 (fragment 1) Phe Phe Gly Ala Val Ile Gly Thr Ile Ala Leu Gly Val Ala Thr Ser 1 5 10 15 Ala Gln Ile Thr Ala Gly Ile Ala Leu Ala Glu Ala Arg Glu Ala Lys 20 25 30 Arg Asp Ile Ala Leu Ile Lys Glu Ser Met Thr Lys Thr His Lys Ser 35 40 45 Ile Glu Leu Leu Gln Asn Ala Val Gly Glu Gln Ile Leu Ala Leu Lys 50 55 60 Thr Leu Gln Asp Phe Val Asn Asp Glu Ile Lys Pro Ala Ile Ser Glu 65 70 75 80 Leu Gly Cys Glu Thr Ala Ala Leu Arg Leu Gly Ile Lys Leu Thr Gln 85 90 95 His Tyr Ser Glu Leu Leu Thr Ala Phe Gly Ser Asn Phe Gly Thr Ile 100 105 110 Gly Glu Lys Ser Leu Thr Leu Gln Ala Leu Ser Ser Leu Tyr Ser Ala 115 120 125 Asn Ile Thr Glu Ile Met Thr Thr Ile Arg Thr Gly Gln Ser Asn Ile 130 135 140 Tyr Asp Val Ile Tyr Thr Glu Gln Ile Lys Gly Thr Val Ile Asp Val 145 150 155 160 Asp Leu Glu Arg Tyr Met Val Thr Leu Ser Val Lys Ile Pro Ile Leu 165 170 175 Ser Glu Val Pro Gly Val Leu Ile His Lys Ala Ser Ser Ile Ser Tyr 180 185 190 Asn Ile Asp Gly Glu Glu Trp Tyr Val Thr Val Pro Ser His Ile Leu 195 200 205 Ser Arg Ala Ser Phe Leu Gly Gly Ala Asp Ile Thr Asp Cys Val Glu 210 215 220 Ser Arg Leu Thr Tyr Ile Cys Pro Arg Asp Pro Ala Gln Leu Ile Pro 225 230 235 240 Asp Ser Gln Gln Lys Cys Ile Leu Gly Asp Thr Thr Arg Cys Pro Val 245 250 255 Thr Lys Val Val Asp Ser Leu Ile Pro Lys Phe Ala Phe Val Asn Gly 260 265 270 Gly Val Val Ala Asn Cys Ile Ala Ser Thr Cys Thr Cys Gly Thr Gly 275 280 285 Arg Arg Pro Ile Ser Gln Asp Arg Ser Lys Gly Val Val Phe Leu Thr 290 295 300 His Asp Asn Cys Gly Leu Ile Gly Val Asn Gly Val Glu Leu Tyr Ala 305 310 315 320 Asn Arg Arg Gly His Asp Ala Thr Trp Gly Val Gln Asn Leu Thr Val 325 330 335 Gly Pro Ala Ile Ala Ile Arg Pro Val Asp Ile Ser Leu Asn Leu Ala 340 345 350 Asp Ala Thr Asn Phe Leu Gln Asp Ser Lys Ala Glu Leu Glu Lys Ala 355 360 365 Arg Lys Ile Leu Ser Glu Val Gly Arg Trp Tyr Asn Ser Arg Glu Thr 370 375 380 Val Ile Thr Ile Ile Val Val Met Val Val Ile Leo Val Val Ile Ile 385 390 395 400 Val Ile Ile Ile Val Leu Tyr Arg Leu Arg Arg 405 410 <210> SEQ ID NO: 15 <211> 91 <223> Fct4 (fragment 2) Gln Ile Pro Arg Asp Arg Leu Ser Asn Ile Gly Val Ile Val Asp Glu 1 5 10 15 Gly Lys Ser Leu Lys Ile Ala Gly Ser His Glu Ser Arg Tyr Ile Val 20 25 30 Leu Ser Leu Val Pro Gly Val Asp Phe Glu Asn Gly Cys Gly Thr Ala 35 40 45 Gln Val Ile Gln Tyr Lys Ser Leu Leu Asn Arg Leu Leu Ile Pro Leu 50 55 60 Arg Asp With Asp To Gln Glu With Thr Val Thr Asp 65 70 75 80 Three Gln Asn Ala Gly Ala Pro Gln Ser Arg 85 90 <210> SEQ ID NO: 16 <211> 25 <223> Fct4 signal peptide MATYIQRVQC ISTSLLVVLT TLVSC 25 <210> SEQ ID NO: 17 <211> 4391 <223> codon-optimized SIV gal-pol nucleic acid sequence (from pGM691) atgggagctg ccacatctgc cctgaataga cggcagctgg accagttcga gaagatcaga 60 ctgcggccca acggcaagaa gaagtaccag atcaagcacc tgatctgggc cggcaagag 120 atggaagat tcggcctgca cgagcggctg ctggaaccg aggaggctg caagagaatt 180 atcgaggtgc tgtaccctct ggaacctacc ggctctgagg gcctgaagtc cctgttcaat 240 ctcgtgtgcg tgctgtactg cctgcacaa gacagaaag tgaggacac cgaagaggcc 300 gtggccacag ttagacagca ctgccacctg gtggaaaaag agaagtccgc cacagagacaca 360 agcagcggcc agaagaagaa cgacaaggga attgctgccc ctcctggcgg cagccagaat 420 tttcctgctc agcagcaggg aaacgcctgg gtgcacgttc cactgagccc tagaacactg 480 aatgcctggg tcaaagccgt ggaagagaag aagtttggcg ccgagatcgt gcccatgttc 540 caggctctgt ctgagggctg caccccttac gacatcaacc agatgctgaa cgtgctggga 600 gatcaccagg gcgctctgca gatcgtgaaa gagatcatca acgaagaggc tgcccagtgg 660 gacgtgacac atccattgcc tgctggacct ctgccagccg gacaactgag agatcctaga 720 ggctctgata tcgccggcac caccagctct gtgcaagagc agctggaatg gatctacacc 780 gccaatccta gagtggacgt gggcgccatc tacagaagat ggatcatcct gggcctgcag 840 aaatgcgtga agatgtacaa ccccgtgtcc gtgctggaca tcagacaggg acccaaagag 900 cccttcaagg actacgtgga ccggttctat aaggccatta gagccgagca ggccagcggc 960 gaagtgaagc agtggatgac agagagcctg ctgatccaga acgccaatcc agactgcaaa 1020 gtgatcctga aaggcctggg catgcacccc acactggaag agatgctgac agcctgtcaa 1080 ggcgttggcg gcccttctta caaagccaaa gtgatggccg agatgatgca gaccatgcag 1140 aaccagaaca tggtgcagca aggcggccct aagacaga ggcctcctct gagatgctac 1200 aactgcggca agttcggcca catgcagaga cagtgtcctg agcctaggaa aacaaaaatgt 1260 ctaaagtgtg gaaaattggg acacctagca aaagactgca ggggacaggt gaatttttta 1320 gggtatggac ggtggatggg ggcaaaaccg agaaattttc ccgccgctac tcttggagcg 1380 gaaccgagtg cgcctcctcc accgagcggc accaccccat acgacccagc aaagaagctc 1440 ctgcagcaat atgcagagaa agggaaaca ctgagggagc aaaagagaa tccaccggca 1500 atgaatccgg attggaccga gggatattct ttgaactccc tctttggaga agaccaataa 1560 agaccgtgta catcgagggc gtgcccatca aggctctgct ggatacaggc gccgacgaca 1620 ccatcatcaa agagaacgac ctgcagctga gcggcccttg gaggcctaag atcattggag 1680 gaatcgggggg aggcctgaac gtcaaagagt acaacgaccg ggaagtgaag atcgagca 1740 agatcctgag gggcacaatc ctgctgggcg ccacacctat caacatcatc ggcagaaatc 1800 tgctggcccc tgccggcgct agactggtta tgggacagct ctctgagaag atccccgtga 1860 cacccgtgaa gctgaaagaa gcgctagag gaccttgtgt gcgacagtgg cctctgagca 1920 agagagat tgaggccctg cagaaatct gtagccagct ggaacagag ggcaagatca 1980 gcagagttgg cggcgagaac gcctacaata cccctatctt ctgcatcaag aaaaggaca 2040 agagccagtg gcggatgctg gtggacttta gagagctgaa caggctacc caggacttct 2100 tcgaggtgca gctgggaatt cctcatcctg ccggcctgcg gaagatgaga cagatcacag 2160 tgctggatgt gggcgacgcc tactacacca tccctctgga cccactc agaagtaca 2220 ccgccttcac aatccccacc gtgacaatc aaggccctgg catcagatac cagttcact 2280 gcctgcctca aggctggaag ggcagcccca ccattttca gatataccgcc gccagcatcc 2340 tggaagaat cagagaac ctgcctgctc tgaccatcgt gcagtacatg gacgatctgt 2400 gggtcggaag ccagagaat gagcacaccc gggacaagct ggtggacag ctgagacaaca 2460 agctgcaggc ctggggccctc gaaacccctg agagaaggt gcagaaagaa cctccttacg 2520 agtggatggg ctacaagctg tggcctcaca agtgggagct gagccggatt cagctcgaag 2580 agaaggacga gtggaccgtg aacgacatcc agaaactcgt gggcaagctg aattgggcag 2640 cccagctgta tcccggcctg aggaccaaga acatctgcaa gctgatccgg ggaaagaaga 2700 acctgctgga actggtcaca tggacacctg aggccgaggc cgaatatgcc gagaatgccg 2760 aaatcctgaa aaccgagcaa gaggggacct actacaagcc tggcattcca atcagagctg 2820 ccgtgcagaa actggaaggc ggccagtggt cctaccagtt taagcaagaa ggccaggtcc 2880 tgaaagtggg caagtacacc aagcagaaga acacccacac caacgagctg aggacactgg 2940 ctggcctggt ccagaaaatc tgcaaagagg ccctggtcat ttggggcatc ctgcctgttc 3000 tggaactgcc cattgagcgg gaagtgtggg aacagtggtg ggccgattac tggcaagtgt 3060 cttggatccc cgagtgggac ttcgtgtcta cccctcctct gctgaaactg tggtacaccc 3120 tgacaaaaga gcccattcct aaagaggacg tctactacgt tgacggcgcc tgcaaccgga 3180 actccaaaga aggcaaggcc ggctacatca gccagtacgg caagcagaga gtggaaaccc 3240 tggaaaacac caccaaccag caggccgagc tgaccgccat taagatggcc ctggagagata 3300 gcggccccaa tgtgaacatc gtgaccgact ctcagtacgc catgggaatc ctgacagccc 3360 agcctacaca gagcgatagc cctctggttg agcagatcat tgccctgatg attcagaagc 3420 agcaaatcta cctgcagtgg gtgcccgctc acaaaggcat cggcggaaac gaagagatcg 3480 ataagctggt gtccaaggga atcagacggg tgctgttcct ggaaaagatt gaagaggccc 3540 aagaggaaca cgagcgctac cacaacaact ggaagaatct ggccgacacc tacggactgc 3600 cccagatcgt ggccaaagaa atcgtggcta tgtgccccaa gtgtcagatc aagggcgaac 3660 ctgtgcacgg ccaagtggat gcttctcctg gcacatggca gatggactgt acccacctgg 3720 aaggcaaagt ggtcatcgtg gctgtgcacg tggcctccgg ctttattgag gccgaagtga 3780 tccccagaga gacaggcaaa gaaaccgcca agttcctgct gaagatcctg tccagatggc 3840 ccatcacaca gctgcacacc gacaacggcc ctaacttcac atctcaagag gtggccgcca 3900 tctgttggtg gggaaagatt gagcacacaa ccggcattcc ctacaatcca cagagccagg 3960 gcagcatcga gtccatgaac aagcagctca aagagattat cggcaagatc cgggacgact gccagtacac agaaacagcc gtgctgatgg cctgtcacat ccacaacttc aagcggaag gcggcatcgg aggacagaca tctgccgaga gactgatcaa tatcatcacc actcagctgg aaatccagca cctccagacc aagatccaga agattctgaa cttccgggtg tactaccgcg agggcagaga tcctgtttgg aaaggcccag cacagctgat ctggaaaggc gaaggtgccg tggtgctgaa ggatggctct gatctgaagg tggtgcccag acggaaggcc aagattatca aggattacga gcccaaacag cgcgtgggca atgaaggcga cgttgagggc acaagaggca 4380. gcgacaattg a <210> SEQ ID NO: 18 <211> 4391 <213> Wild-type Simian immunodeficiency virus gagpole atgggggcgg ctacctcagc actaatagg agacaattag accaatttga gaaatacga cttcgcccga acggaaga aaagtaccaa attaacatt taatatgggc aggcaaggag atggagcgct tcggcctcca tgagaggttg ttggagacag aggaggggtg taaaagaatc atagaagtcc tctaccccct agaaccaca ggatcggagg gcttaaaag tctgttcaat 240 cttgtgtgcg tactatattg cttgcacaag gacagaaag tgaagacac agaggaagca 300 gtagcacag taagacaca ctgccatcta gtggaaaag aaaaagtgc aacagagaca 360 tctagtggac aaagaaaaa tgacaaggga atagcagcgc cacctggtgg cagtcagaat 420 tttccagcgc aaaacagg aaatgcctgg gtacatgtac ccttgtcacc gcgcacctta 480 aatgcgtggg taaaagcagt agaggagaaaaatttggag cagaatagt acccatgttt 540 caagccctat cagaaggctg cacacctat gataatc agatgcttaa tgtgctagga 600 gatcatcaag gggcattaca atagtgaaa gagatcatta atgagaagc agcccagtgg 660 gatgtaacac acccactacc cgcaggaccc ctaccagcag gagactcag ggaccctcgc 720 ggctcagata tagcagggac caccagctca gtacagaac agttagaatg gatctatact 780 gctaaccccc gggtagatgt aggtgccatc taccggagat ggattattct aggacttca 840 aagtgtgtca aaatgtacaa cccagtatca gtcctagaca ttaggcaggg acctaaagg 900 cccttcaagg attatgtgga cagattttac aaggcaatta gagcagaaca agcctcaggg 960 gaagtgaaac aatggatgac agaatcatta ctcattcaaa atgctaatcc agattgtaag 1020 gtcatcctga agggcctagg aatgcacccc acccttgaag aaatgttaac ggcttgtcag 1080 ggggtaggag gcccaagcta caaagcaaaa gtaatggcag aaatgatgca gaccatgcaa 1140 aatcaaaaca tggtgcagca gggaggtcca aaaagacaaa gacccccact aagatgttat 1200 aattgtggaa aatttggcca tatgcaaaga caatgtccgg aaccaaggaa aacaaaatgt 1260 ctaaagtgtg gaaaattggg acacctagca aaagactgca ggggacaggt gaatttttta 1320 gggtatggac ggtggatggg ggcaaaaccg agaaattttc ccgccgctac tcttggagcg 1380 gaaccgagtg cgcctcctcc accgagcggc accacccat acgacccagc aaagaagctc 1440 ctgcagcaat atgcagagaa agggaaacaa ctgagggagc aaaagaggaa tccaccggca 1500 atgaatccgg attggaccga gggatattct ttgaactccc tctttggaga agccaataa 1560 agacagtgta tatagaaggg gtccccatta aggcactgct agacacaggg gcagatgaca 1620 ccatattaa agaaaatgat ttacaattat caggtccatg gagacccaaa attatagggg 1680 gcataggagg aggccttaat gtaaaagaat ataacgacag ggaagtaaaa atagagata 1740 aaattttgag aggaacaata ttgttaggag shaacccat tatataata ggtagaaatt 1800 tgctggcccc ggcaggtgcc cggttagtaa tgggacaatt atcagaaaaa attcctgtca 1860 cacctgtcaa attgaggaa ggggctcggg gaccctgtgt aagacaatgg cctctctcta 1920 agagagat tgaagcttta caggaat gttcccatt agagcaggaa ggaaaatca 1980 gtagagtagg aggaaaat gcatacaata cccaatatt ttgcataag agaaggaca 2040 aatcccagtg gaggatgcta gtagacttta gagagttaa taaggcacc gaggattctct 2100 ttgaagtgca attaggata ccccaccag caggattag aagatgaga attaggatacag 2160 ttttagatgt aggacgcc tattattcca taccattgga tccaaatttt aggaaata 2220 ctgcttttac tattcccaca gtgaataatc agggacccgg gattaggtat cattcact 2280 gtctcccgca agggtggaaa ggatctccta caatcttcca aaatacagca gcatccattt 2340 tggaggagat aaaagaaac ttgccagcac taaccattgt acaatacatg gatgatttat 2400 gggtaggttc tcagaaat gacacaccc atgacaatt aggagacag tcagaaa 2460 attackacaagc ctggggct gaaaccccag aaagaaggt gcaaaagaa ccaccttatg 2520 agtggatgggg atacaactt tggcctcaca aatgggaact agcagaata siactggagg 2580 aaaagatga atggactgtc atgacatcc agaagttagt tgggaacta aattggggcag 2640 cacaattgta tccaggtctt aggaccaga atatatgcaa gttattaga ggaaagaaaaa 2700 atctgttaga gctagtgact tggacacctg aggcagaagc tgaatatgca gaaaatgcag 2760 agattcttta aagaacag gaggaacct attackaaacc aggatacct attaggggcag 2820 cagtacagaa attggaagga ggacagtgga gttaccaatt caacagaa ggacaagtct 2880 tgaaagtagg aaatacacc aaagcaaaga acacccatac aaatgaactt cgcacattag 2940 ctggtttagt gcagaagatt tgcaagag ctctagttat ttggggata ttaccagttc 3000 tagactccc gatagaaga gaggtatggg aacaatggtg ggcggattac tggcaggtaa 3060 gctggattcc cgaatgggat ttgtcagca cccaccttt gctcaacta tggtacacat 3120 taacaaaga acccatacccc aaggaggacg tttactatgt agatggagca tgcaacagaa 3180 attcaaaga aggaaagca ggatacatct cacaatacgg aaaacagaga gtagaaacat 3240 taggaaacac taccaatcag taccagaat aaaaatggct ttggagaca 3300 gtgggcctaa tgtgaacata gtaacagact ctcaatagc aatgggaatt ttgacagcac 3360 aaccacaca aagtgattca ccattagtag agcaattat agccttaatg atacaaagc 3420 aaaaata tttgcagtgg gtaccagcac aaaggaat agggaggaat gaggagatag 3480 attaattagt gagtaaggc attagaagg ttttattctt agaaaaaata gagaagctc 3540 agcagataca tatgggcttc 3600 cacaatagt agcaaagag atagtggcca tgtgtccaaa atgtcagata aagggagaac 3660 cagtgcatgg acagtggat gcctcacctg gaacatggca catggattgt actcatctag 3720 aaggaaaagt agtcatagtt gcggtccatg tagccagtgg attcatagaa gcagaagtca 3780 tacctaggga aacaggaaaa gaacggcaa agttcttatt aaaaatactg agtagatggc 3840 ctataacaca gttacacaca gatagggc ctactttac ctcccaagaa gtggcagcaa 3900 tatgttgtg gggaaaaatt gaacataca caggtatacc atattacccc caatctcaag 3960 gatchataga aagcatgac aaacattaa aagataat tgggaaata agagatgatt 4020 gccaatatac agagacagca gtactgatgg cttgccatat tcacaattt aaagaaagg 4080 gaggaatagg gggacagact tcaxagaga gaccaattaa father acashaattag 4140 aaatacaaca tttacaacc aaattcaa aaattttaaa ttttagagtc tactacagag 4200 aagggagaga ccctgtgtgg aaggaccag cacattaat ctggaaggg gaoggagcag 4260 tggtcctcaa ggacggaagt gacctaagg ttgtaccaag aaggaagct aaaattatta 4320 aggattga acccaaaaa agatgggta atgagggtga cgtggaaggt accagggat 4380 ctgataacta a 4391 <210> SEQ ID NO: 19 <211> 10536 <223> pGM830 ggtacctcaa tattggccat tagccatatt attcattggt tatatagcat aaatcaatat 60 tggctattgg ccattgcata cgttgtatct atatcataat atgtacattt atattggctc 120 atgtccaata tgaccgccat gttggcattg attattgact agttattaat agtaatcaat 180 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 240 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgtgt 300 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt attacggta 360 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtccgcccc ctattgacgt 420 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttac gggactttcc 480 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca 540 gtacaccaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat 600 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa 660 caactgcgat cgcccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc 720 tatataagca gagctcgctg gcttgtaact cagtctctta ctaggagacc agcttgagcc 780 tgggtgttcg ctggtagcc taacctggtt ggccaccagg ggtaaggact ccttggctta 840 gaaagctaat aaacttgcct gcattagagc ttatctgagt caagtgtcct cattgacgcc 900 tcactctctt gaacgggaat cttccttact gggttctctc tctgacccag gcgagagaaa 960 ctccagcagt ggcgcccgaa cagggacttg agtgagagtg taggcacgta cagctgagaa 1020 ggcgtcggac gcgaaggaag cgcggggtgc gacgcgacca agaaggagac ttggtgagta 1080 ggcttctcga gtgccgggaa aaagctcgag cctagttaga ggactaggag aggccgtagc 1140 cgtaactact ctgggcaagt agggcaggcg gtgggtacgc aattggggc ggctacctca 1200 gcactaaata ggagacaatt agaccaattt gagaaaatac gacttcgccc gaacggaaag 1260 aaaaagtacc aaattaaaca tttaatattg ggcaggcaag gagattggag cgcttcggcc 1320 tccatgagag gttgttggag acagaggagg ggtgtaaaag aatcatagaa gtcctctacc 1380 ccctagaacc aacaggatcg gagggcttaa aaagtctgtt caatcttgtg tgcgtgctat 1440 attgcttgca hyagaaag aaagtgaaag ahaaaag aggagtagca hyagaaag 1500 aacactgcca tctagtggaa aaaaaaaa gtgcaacaga gatactgt ggacaaaga 1560 aaaatgacaa gggaatagca gcgccacctg gtggcagtca gatttttcca gcgcacaac 1620 aaggaaattg cctgggtaca tgtaccttg tcaccgcgca ccttaaatgc gtgggtaaaa 1680 gcagtagagg agaaaaaatt tggagcagaa atagtaccca tgtttcaagc cctatcgcct 1740 1800 cagcggcgac agccctgacg gtccagtctc agcattttgct tgctgggata ctgcagcagc 1860 agagaatct gctggcggct gtggaggctc aacagcagat gttgaagctg accattttgg 1920 gtgttaaaaa cctcaatgcc cgcgtcacag cccttgagaa gtacctagag gatcaggcac 1980 gactaaactc ctgggggtgc gcatggaaac aagtatgtca taccacagtg gagtggccct 2040 ggacaatcg gactccggat tggcaaata agacttggtt ggagtgggaa agacaatag 2100 ctgatttgga aagcacatt frequency taggaggc tgagaaa frequency 2160 atctagatgc ctacagaag ttaactagtt ggtcagattt ctggtcttgg ttcgatttct 2220 caaaatggct taacatttta aaaagggat ttttagtaat aggaggata attaggtta 2280 gattacttta cacagtatat ggatgtatag tgaggttag gcaggtat gttccctctat 2340 ctccacagat ccatataag cggcaatttt aaaagaaagg gaggaatagg gggacagact 2400 tcagcagaga gactattaa tataaca acacaattag aatacaca tttacaacc 2460 aaaattcaa aaattttaaa ttttagagcc gcggagatct gttacataac tttaggtaaa 2520 tggcctgcct ggctgactgc ccaatgaccc ctgcccaatg atgtcaataa tgatgtatgt 2580 tcccatgtaa tgccaatagg gactttccat tgatgtcaat gggtggagta tttatggtaa 2640 ctgcccactt ggcagtacat caagtgtatc atatgccaag tatgccccct attgatgtca 2700 atgatggtaa atggcctgcc tggcattg cccagtacat gaccttatgg gactttccta 2760 cttggcagta catctatgta ttagtcattg ctattaccat gggaattcac tagtggagaa 2820 gagcatgctt gagggctgag tgcccctcag tggcagaga gcacatggcc cacagtccct 2880 gagaagttgg ggggaggggt gggcaattga actggtgcct agaaggtg gggcttgggt 2940 aaactgggaa agtgatgtgg tgtactggct ccacctttt ccccagggtg ggggagaacc 3000 atatataagt gcagtagtct ctgtgaaacat tcaagcttct gccttctccc tcctgtgagt 3060 ttgctagcca ccatgcagag aagccctctg gagaaggcct ctgtggtgag caagctgttc 3120 3180 atctaccaga tcccctctgt ggactctgct gacaacctgt ctgagaagct ggagagggag 3240 tgggatagag agctggccag caagaagaac cccaagctga tcaatgccct gaggagatgc 3300 ttcttctgga gattcatgtt ctatggcatc ttcctgtacc tgggggaagt gaccaaggct 3360 gtgcagcctc tgctgctggg cagaatcatt gccagctatg accctgacaa caaggaggag 3420 aggagcattg ccatctacct gggcattggc ctgtgcctgc tgttcattgt gaggaccctg 3480 ctgctgcacc ctgccatctt tggcctgcac cacattggca tgcagatgag gattgccatg 3540 ttcagcctga tctacaagaa aaccctgaag ctgtccagca gagtgctgga caagatcagc 3600 attggccagc tggtgagcct gctgagcaac aacctgaaca agtttgatga gggcctggcc ctggcccact ttgtgtggat tgcccctctg caggtggccc tgctgatggg cctgatttgg 3720 gagctgctgc aggcctctgc cttttgtggc ctgggcttcc tgattgtgct ggccctgttt 3780 caggctggcc tgggcaggat gatgatgaag tacagggacc agagggcagg caagatcagt gagaggctgg tgatcacctc tgagatgatt gagacatcc agtctgtgaa ggcctactgt tgggaggag ctatggaga gatgattga aacctgaggc agacagagct gagctgacc aggaaggctg cctatgtgag atacttcaac agctctgcct tcttcttctc tggcttcttt gtggtgttcc tgtctgtgct gccctatgcc ctgatcaagg ggatcatcct gagaaagatt 4080 ttcaccacca tcagcttctg cattgtgctg aggatggctg tgaccagaca gttcccctgg gctgtgcaga cctggtatga cagcctgggg gccatcaaca agatccagga cttcctgcag aagcaggagt acaagaccct ggagtacaac ctgaccacca cagaagtggt gatggagaat gtgacagcct tctgggagga gggctttggg gagctgtttg agaaggccaa gcagaacaac aacaacagaa agaccagcaa tggggatgac tccctgttct tctccaactt ctccctgctg 4380 ggcacacctg tgctgaagga catcaacttc aagattgaga gggggcagct gctggctgtg 4440 gctggatcta caggggctgg caagaccagc ctgctgatga tgatcatggg ggagctgggag 4500 ccttctgagg gcaagatcaa gcactctggc aggatcagct tttgcagcca gttcagctgg 4560 atcatgcctg gcaccatcaa ggagaacaatc atctttggag tgagctatga tgagtacaga 4620 tacaggagtg tgatcaaggc ctgccagctg gaggagaca tcagcaagtt tgctgagaag 4680 gacaacattg tgctggggga gggaggcatt acactgtctg gggggccagag agccagaatc 4740 agcctggcca gggctgtgta caaggatgct gacctgtacc tgctggactc cccctttggc 4800 tacctggatg tgctgacaga gaagggatt tttgagagct gtgtgtgcaa gctgatggcc 4860 aacaagacca gaatcctggt gaccagcaag atggagcacc tgagaaggc tgacaagatc 4920 ctgatcctgc atgaggcag cagctacttc tatgggacct tctctgagct gcagaacctg 4980 cagcctgact tcagctctaa gctgatgggc tgtgacagct ttgaccagtt ctctgctgag 5040 aggaggaca gcatcctgac aggaccctg aggattca gcctggaggg agatgcccct gtgagctgga cagagaccaa gaagcagagc ttcaagcaga caggggagtt tggggagaag aggaagaact ccatcctgaa ccccatcaac agcatcagga agttcagcat tgtgcagaaa acccccctgc agatgaatgg cattgagga gattctgatg agcccctgga gaggagactg agcctggtgc ctgattctga gcagggagag gccatcctgc ctaggatctc tgtgatcagc 5340 acaggcccta cactgcaggc cagaaggagg cagtctgtgc tgaacctgat gacccactct gtgaaccagg gccagaacat ccacaggaa accacagcct ccaccagga agtgagcctg gcccctcagg ccaatctgac agagctggac atctacagca ggaggctgtc tcaggagac ggcctggaga tttctgagga gatcaatgag gaggacctga aagagtgctt ctttgatgac 5580 atggagagca tccctgctgt gaccacctgg aacacctacc tgagatacat cacagtgcac aagagcctga tctttgtgct gatctggtgc ctggtgatct tcctggctga agtggctgcc 5700 tctctggtgg tgctgtggct gctgggaaac accccactgc aggacaaggg caacagcacc cacagcagga acaacagcta tgctgtgatc atcacctcca cctccagcta ctatgtgttc 5820 tacatctatg tgggagtggc tgataccctg ctggctatgg gcttctttag aggcctgccc 5880 ctggtgcaca cactgatcac agtgagcaag atcctccacc acaagatgct gcactctgtg 5940 ctgcaggctc ctatgagcac cctgaatacc ctgaaggctg ggggcatcct gaacagattc 6000 tccaaggata ttgccatcct ggatgacctg ctgcctctca ccatctttga cttcatccag 6060 ctgctgctga ttgtgattgg ggccattgct gtggtggcag tgctgcagcc ctacatcttt 6120 gtggccacag tgcctgtgat tgtggccttc atcatgctga gggcctactt tctgcagacc 6180 tcccagcagc tgaagcagct ggagtctgag ggcagaagcc ccatcttcac ccacctggtg 6240 acaagcctga agggcctgtg gaccctgaga gcctttggca ggcagcccta ctttgagacc 6300 ctgttccaca aggccctgaa cctgcacaca gccaactggt tcctctacct gtccaccctg 6360 agatggttcc agatgagaat tgagatgatc tttgtcatct tcttcattgc tgtgaccttc 6420 atcagcattc tgaccacagg agagggagag ggcagagtgg gcattatcct gaccctggcc 6480 atgaacatca tgagcacact gcagtgggca gtgaacagca gcattgatgt ggacagcctg atgaggagtg tgagcagagt gttcaagttc attgatatgc ccacagaggg caagcctacc aagagcacca agccctacaa gaatggccag ctgagcaaag tgatgatcat tgagaacagc catgtgaaga aggatgatat ctggcccagt ggaggccaga tgacagtgaa ggacctgaca 6720 gccaagtaca cagagggggg caatgctatc ctggagaca tctccttcag catctcccct ggccagagag tgggactgct gggagaca ggctctggca agtctaccct gctgtctgcc 6840. ttcctgaggc tgctgaacac agagggagag atccagattg atggagtgtc ctgggacagc 6900 atcacactgc agcagtggag gaaggcctttt ggtgtgatcc cccagaaagt gttcatcttc 6960. agtggcacct tcaggaga cctggacccc tatgagcagt ggtctgacca ggagatttgg aaagtggctg atgaagtggg cctgagaagt gtgattgagc agttccctgg caagctggac 7080. tttgtcctgg tggatggggg ctgtgtgctg agccatggcc acaagcagct gatgtgcctg 7140 gccagatcag tgctgagcaa ggccagatc ctgctgctgg atgagccttc tgcccacctg 7200 gatcctgtga cctaccagat catcaggagg accctcaagc aggcctttgc tgactgcaca 7260 gtcatcctgt gtgagcacag gattgaggcc atgctggagt gccagcagtt cctggtgatt 7320 gaggagaaca aagtgaggca gtatgacagc atccagaagc tgctgaatga gaggagcctg 7380 ttcaggcagg ccatcagccc ctctgataga gtgaagctgt tcccccacag gaacagctcc 7440 aagtgcaaga gcaagcccca gattgctgcc ctgaaggagg agacagagga ggaagtgcag 7500 gacaccaggc tgtgagggcc caatcaacct ctggattaca aaatttgtga aagattgact 7560 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg 7620 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg 7680 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg 7740 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg 7800 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc 7860 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca 7920 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc 7980 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct 8040 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc 8100 gcctccccgc aagcttcgca ctttttaaaa gaaaagggag gactggatgg gatttattac 8160 tccgatagga cgctggcttg taactcagtc tcttactagg agaccagctt gagcctgggt 8220 gttcgctggt tagcctaacc tggttggcca ccaggggtaa ggactccttg gcttagaaag 8280 ctaataaact tgcctgcatt agagctctta cgcgtcccgg gctcgagatc cgcatctcaa 8340 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag 8400 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc 8460 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt 8520 ttgcaaaaag ctaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc 8580 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc 8640 atcaatgtat cttatcatgt ctgtccgctt cctcgctcac tgactcgctg cgctcggtcg 8700 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 8760 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 8820 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 8880 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 8940 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 9000 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 9060 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 9120 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 9180 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 9240 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct 9300 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 9360 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 9420 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 9480 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 9540 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 9600 gttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa 9660 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc 9720 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac 9780 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga 9840 ctgaatccgg tgagaatggc aacagcttat gcatttcttt ccagacttgt tcaacaggcc 9900 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt 9960 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg 10020 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat 10080 attcttctaa tacctggaat gctgtttttc cggggatcgc agtggtgagt aaccatgcat 10140 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt 10200 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa 10260 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga 10320 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg 10380 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta 10440 tgtaagcaga cagttttatt gttcatgatg atatattttt atcttgtgca atgtaacatc 10500 agagattttg agacacaaca attggtcgac ggatcc 10536 <210> SEQ ID NO: 20 <211> 9064 <223> pGM691 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 60 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 120 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 180 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 240 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 300 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 360 tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc 420 ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg 480 gggcgggggg gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg 540 gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt 600 ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag 660 tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc 720 ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg 780 gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc 840 ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggggt gcgtgcgtgt 900 gtgtgtgcgt ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt gagcgctgcg 960 ggcgcggcgc ggggctttgt gcgctccgca gtgtgcgcga ggggagcgcg gccgggggcg 1020 gtgccccgcg gtgcgggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1080 tgggggggtg agcagggggt gtgggcgcgt cggtcgggct gcaacccccc ctgcaccccc 1140 ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg 1200 cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc 1260 cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg cccccggagc gccggcggct 1320 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1380 gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct 1440 ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc 1500 ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca gcctcggggc tgtccgcggg 1560 gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg 1620 gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg 1680 gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaattgctc gagccaccat 1740 gggagctgcc acatctgccc tgaatagacg gcagctggac cagttcgaga agatcagact 1800 gcggcccaac ggcaagaaga agtaccagat caagcacctg atctgggccg gcaaagagat 1860 ggaaagattc ggcctgcacg agcggctgct ggaaaccgag gaaggctgca agaaattat 1920 cgaggtgctg taccctctgg aacctaccgg ctctgagggc ctgaagtccc tgttcaatct 1980 cgtgtgcgtg ctgtactgcc tgcacaaaga acagaaagtg aaggacaccg aagaggccgt 2040 ggccacagtt agacagcact gccacctggt ggaaaaagag aagtccgcca cagagacaag 2100 cagcggccag aagaagaacg acaagggaat tgctgcccct cctggcggca gccagaattt 2160 tcctgctcag cagcaggaa acgcctgggt gcacgttcca ctgagcccta gaacactgaa 2220 tgcctgggtc aaagccgtgg aagagaagaa gtttggcgcc gagatcgtgc ccatgttcca 2280 ggctctgtct gagggctgca ccccttacga catcaaccag atgctgaacg tgctgggaga 2340 tcaccagggc gctctgcaga tcgtgaaaga gatcatcaac gaagaggctg cccagtggga 2400 cgtgacacat ccattgcctg ctggacctct gccagccgga caactgagag atcctagagg 2460 ctctgatatc gccggcacca ccagctctgt gcaagagcag ctggaatgga tctacaccgc 2520 caatcctaga gtggacgtgg gcgccatcta cagaagatgg atcatcctgg gcctgcagaa 2580 atgcgtgaag atgtacaacc ccgtgtccgt gctggacatc agacagggac ccaaagagcc 2640 cttcaaggac tacgtggacc ggttctataa ggccattaga gccgagcagg ccagcggcga 2700 agtgaagcag tggatgacag agagcctgct gatccagaac gccaatccag actgcaaagt 2760 gatcctgaaa ggcctgggca tgcaccccac actggaagag atgctgacag cctgtcaagg 2820 cgttggcggc ccttcttaca aagccaaagt gatggccgag atgatgcaga ccatgcagaa 2880 ccagaacatg gtgcagcaag gcggccctaa gagacagagg cctcctctga gatgctacaa 2940 ctgcggcaag ttcggccaca tgcagagaca gtgtcctgag cctaggaaaa caaaatgtct 3000 aaagtgtgga aaattgggac acctagcaaa agactgcagg ggacaggtga attttttagg 3060 gtatggacgg tggatgggg caaaaccgag aaattttccc gccgctactc ttggagcgga 3120 accgagtgcg cctctcccac cgagcggcac cacccatac gacccagcaa agaagctcct 3180 gcagcaatat gcagagaaag ggaaacaact gagggagcaa aaggagaatc caccggcaat 3240 3300 accgtgtaca tcgagggcgt gcccatcaag gctctgctgg atacaggcgc cgacgacacc 3360 atcatcaaag agaacgacct gcagctgagc ggcccttgga ggcctaagat cattggagga 3420 atcggcggag gcctgaacgt caaagagtac aacgaccggg aagtgaagat cgagcaag 3480 atcctgaggg gcacaatcct gctggggcgcc acacctatca acatcatcgg cagaaatctg 3540 ctggcccctg ccggcgctag actggttatg ggacagctct ctgagaagat ccccgtgaca 3600 cccgtgaagc tgaaagaagg cgctagagga ccttgtgtgc gacagtggcc tctgagcaaa 3660 gagaattg aggccctgca agaaatctgt agccagctgg aacaagaggg caagatcagc 3720 agagttggcg gcgagaacgc ctacaatacc cctatcttct gcatcaagaa aaaggacaag 3780 agccagtggc ggatgctggt ggactttaga gagctgaaca aggctaccca ggacttcttc 3840 gaggtgcagc tgggaattcc tcatcctgcc ggcctgcgga agatgagaca gatcacagtg 3900 ctggatgtgg gcgacgccta ctacagcatc cctctggacc ccaacttcag aaagtacacc 3960 gccttcaa tccccaccgt gaaaatcaa ggccctggca tcagatacca gttcaactgc 4020 ctgcctcaag gctgggaaggg cagcccccacc attttcaga ataccgccgc cagcatcctg 4080 gaagaaatca agaaacct gcctgctctg accatcgtgc agtacatgga cgatctgtgg 4140 gtcggaagcc aagagaatga gcacacccac gacaagctgg tggaacagct gagacaaag 4200 ctgcaggcct ggggcctcga aacccctgag aagaaggtgc agaaagaacc tccttacgag 4260 tggatgggct acaagctgtg gcctcacaag tgggagctga gccggattca gctcgaagag 4320 areacgagt ggaccgtgaa cgacatccag aaactcgtgg gcaagctgaa ttgggcagcc 4380 cagctgtatc ccggcctgag gaccaagaac atctgcaagc tgatccgggg aaagaaac 4440 ctgctgggaac tggtcacatg gacacctgag gccgaggccg aatatgccga gaatgccgaa 4500 atcctgaaaa ccgagcaaga ggggacctac tacaagcctg gcattccaat cagagctgcc 4560 gtgcagaaac tggaggcgg ccagtggtcc taccagtttta agcaagagg ccaggtcctg 4620 aaagtgggca agtacaccaa gcagagaac acccacca acgagctgag vakactggct 4680 ggcctgtcc agaaatctg caaagggcc ctggtcattt ggggcatct gcctgttctg 4740 gaactgccca ttgagcggga agtgtgggaa cagtggtggg ccgattactg gcaagtgtct 4800 ...

Claims

1. 1. A retroviral vector comprising a modified retroviral RNA sequence, said modified retroviral RNA sequence having a sequence similar to that of the unmodified retroviral RNA sequence from which it is derived, said sequence being: (i) a codon substitution; and (ii) contains a reduced number of retroviral open reading frames (ORFs); (a) the retroviral RNA sequence comprises a promoter and a transgene; and (b) the retroviral vector is pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus; The retroviral vector.

2. The modified retroviral RNA sequence is compared to the unmodified retroviral RNA sequence from which the modified retroviral RNA sequence is derived, (a) one or more retroviral ORFs 5' of the promoter: (b) one or more retroviral ORFs encoding a peptide ≥ 100 amino acids in length; (c) one or more retroviral ORFs contained in the partial RRE sequence; and / or (d) one or more retroviral ORFs encoded within the partial Gag sequence; The retroviral vector of claim 1, which lacks:

3. The retroviral vector according to claim 1 or 2, wherein the respiratory paramyxovirus is a Sendai virus.

4. 4. The retroviral vector of claim 1, wherein the promoter is selected from the group consisting of a hybrid human CMV enhancer / EF1a (hCEF) promoter, a cytomegalovirus (CMV) promoter, and an elongation factor 1a (EF1a) promoter, and optionally the retroviral vector comprises a hybrid human CMV enhancer / EF1a (hCEF) promoter.

5. The introduced gene is a) CFTR, ABCA3, DNAH5, DNAH11, DNAI1, and DNAI2; or b) secreted therapeutic proteins, optionally alpha-1 antitrypsin (A1AT), factor VIII, surfactant protein B (SFTPB), factor VII, factor IX, factor X, factor XI, von Willebrand factor, granulocyte-macrophage colony-stimulating factor (GM-CSF) and monoclonal antibodies against infectious agents; The retroviral vector according to any one of claims 1 to 4, which is selected from the group consisting of:

6. The introduced gene is a) CFTR; b) A1AT; or c) FVIII The retroviral vector according to any one of claims 1 to 5, which encodes:

7. a) the promoter is the hCEF promoter and the transgene encodes CFTR; b) the promoter is the hCEF promoter and the transgene encodes A1AT; or c) the promoter is a hCEF or CMV promoter and the transgene encodes FVIII; The retroviral vector according to any one of claims 1 to 6.

8. 8. The retroviral vector of any one of claims 1 to 7, which is a lentiviral vector; optionally, the lentiviral vector is selected from the group consisting of a simian immunodeficiency virus (SIV) vector, a human immunodeficiency virus (HIV) vector, a feline immunodeficiency virus (FIV) vector, an equine infectious anemia virus (EIAV) vector, and a Visna / Maedi virus vector.

9. The retroviral vector according to any one of claims 1 to 8, which is an SIV vector.

10. 10. The retroviral vector of any one of claims 1 to 9, wherein the modified retroviral RNA sequence comprises or consists of a nucleic acid sequence that is (i) less than 9,000 bases in length; and (ii) has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% identity to SEQ ID NO:

1.

11. 11. The retroviral vector of claim 10, wherein the modified retroviral RNA sequence comprises or consists of the nucleic acid sequence of SEQ ID NO: 1, preferably wherein the modified retroviral RNA sequence consists of the nucleic acid sequence of SEQ ID NO:

1.

12. (a) a p17 protein comprising or consisting of an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity to SEQ ID NO:2; (b) a p24 protein comprising or consisting of an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity to SEQ ID NO:3; (c) a p8 protein comprising or consisting of an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity to SEQ ID NO:4; (d) a protease comprising or consisting of an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or at most 100% sequence identity to SEQ ID NO:5; (e) a p51 protein comprising or consisting of an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity to SEQ ID NO: 6; (f) a p15 protein comprising or consisting of an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity to SEQ ID NO: 7; and (g) a p31 protein comprising or consisting of an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity to SEQ ID NO:

8. further comprising one or more of: optionally including each of (a) through (g); The retroviral vector according to any one of claims 1 to 11.

13. (a) a Gag protein comprising or consisting of an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity to SEQ ID NO: 9; and / or (b) a Pol protein comprising or consisting of an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or at most 100% sequence identity to SEQ ID NO:

10. The retroviral vector according to any one of claims 1 to 12, further comprising one or more of the following:

14. The F protein optionally comprises: (a) comprising or consisting of an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity to SEQ ID NO: 12 or 13; and / or (b) cleaved into two subunits, optionally wherein the first subunit comprises or consists of an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity to SEQ ID NO: 14, and the second subunit comprises or consists of an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity to SEQ ID NO: 15; The retroviral vector according to any one of claims 1 to 13, which is an Fct4 protein.

15. An SIV vector pseudotyped with the hemagglutinin-neuraminidase (HN) and fusion (F) proteins of Sendai virus, (a) the SIV vector comprises a modified retroviral RNA sequence comprising or consisting of the nucleic acid sequence of SEQ ID NO:1, preferably the modified retroviral RNA sequence consists of the nucleic acid sequence of SEQ ID NO:1; and (b) the F protein comprises a first subunit comprising or consisting of the amino acid sequence of SEQ ID NO: 14, and a second subunit comprising or consisting of the amino acid sequence of SEQ ID NO: 15; The SIV vector.

16. (a) a p17 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 2; (b) a p24 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 3; (c) a p8 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 4; (d) a protease comprising or consisting of the amino acid sequence of SEQ ID NO: 5; (e) a p51 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 6; (f) a p15 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 7; (g) a p31 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 8; (h) a Gag protein comprising or consisting of the amino acid sequence of SEQ ID NO: 9; and / or (i) a Pol protein comprising or consisting of the amino acid sequence of SEQ ID NO:

10. further comprising one or more of: optionally including each of (a) through (g); The SIV vector of claim 15.

17. A method for producing a retroviral vector as defined in any one of claims 1 to 16, comprising the steps of: a) growing cells in suspension; b) transfecting said cells with one or more plasmids; c) adding a nuclease; d) harvesting the lentivirus; e) adding trypsin or an enzyme with the same cleavage specificity; and f) Purification step The method comprising:

18. 18. The method of claim 17, wherein steps (a) through (f) are performed sequentially.

19. 19. The method of claim 17 or 18, wherein the cells are HEK293T or 293T / 17 cells.

20. The method according to any one of claims 17 to 19, wherein the nuclease is added at a stage before collection.

21. The method according to any of claims 17 to 20, wherein the addition of trypsin or an enzyme with the same cleavage specificity is a post-harvesting step.

22. The method of any of claims 17 to 21, wherein the purification step comprises a chromatography step.

23. A composition comprising a retroviral vector as defined in any one of claims 1 to 16 and a pharmaceutically acceptable excipient or diluent.

24. 24. The composition of claim 23, formulated for administration to the lungs; optionally, said administration is by intratracheal or intranasal instillation, aerosol delivery, intravenous injection, or direct injection into the lungs.

25. A retroviral vector as defined in any one of claims 1 to 16 for use in a method of therapy.

26. A method for treating a disease, comprising the step of administering to a subject in need thereof a retroviral vector as defined in any one of claims 1 to 16.

27. 27. A retroviral vector for use according to claim 25 or a method of treatment according to claim 26, wherein the disease to be treated is a pulmonary disease, preferably cystic fibrosis.

Images