Retroviral vectors

Abstract

The present invention relates to a retroviral vector, in particular a lentiviral vector, comprising a modified retroviral RNA sequence substituted with a codon and comprising a reduced number of retroviral open reading frames, a method for the production thereof, and uses thereof, and wherein the retroviral vector is pseudo-modeled by hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxovirus.

Description

Technical Field

[0001] This invention relates to retroviral vectors, particularly lentiviral vectors, comprising a modified retroviral RNA sequence having codon substitutions and comprising a reduced number of retroviral open reading frames, wherein the retroviral vector is pseudo-patterned with hemagglutinin-neuronalase (HN) and fusion (F) proteins from respiratory paramyxoviruses; a method for manufacturing the vector; and its use. Background Technology

[0002] Retroviruses belong to the family Retroviridae, which encodes the enzyme reverse transcriptase. Lentivirals are a genus within the Retroviridae family and are characterized by a long latency period. Retroviruses, and especially lentiviruses, can deliver large amounts of viral RNA into the DNA of host cells and possess the unique ability to infect non-dividing cells, making them one of the most effective gene delivery vectors.

[0003] Pseudo-patterning is a method for generating combinations of viruses or viral vectors with foreign viral mantle proteins. Therefore, foreign viral mantle proteins can be used to alter the host orientation of viral particles or increase / decrease stability. For example, pseudo-patterning allows us to specify the characteristics of the mantle protein. A protein commonly used for pseudo-patterning retroviral and lentiviral vectors is glycoprotein G of vesicular stomatitis virus (VSV), abbreviated as VSV-G.

[0004] Lentiviral vectors, especially those derived from HIV-1, are widely studied and commonly used. The evolution of the lentiviral vector backbone and the virus's ability to deliver recombinant DNA molecules (transgenic genes) into target cells have enabled their use in numerous applications. Two potential applications of viral vectors include restoring functional genes in gene therapy and producing recombinant proteins in vitro.

[0005] When designing retroviral / lentiviral vectors suitable for gene delivery, a key driving factor is to make the vector as safe as possible for patients. A second key driving factor is the need to produce sufficient quantities of vectors not only to treat individual patients but also to make the therapy more widely available clinically to all patients who could benefit from it. These two driving factors can be conflicting, as modifications to improve vector safety are often associated with reduced yields during vector production.

[0006] One example of a clinical setting that would benefit from gene transfer to the airway epithelium is the treatment of cystic fibrosis (CF). CF is a fatal genetic disorder caused by mutations in the gene for the CF transmembrane transport regulator protein (CFTR), which acts as a chloride ion channel in airway epithelial cells. CF is characterized by recurrent chest infections, increased airway secretions, and eventual respiratory failure. In the UK, the current median age of death is approximately 25 years. For most genotypes, there are no treatments targeting the underlying deficiency; current symptom-relieving treatments require several hours of self-administration and therapy daily. Unlike small molecule drugs, gene therapy is independent of the CFTR mutation class and is therefore applicable to all affected individuals with CF. However, to date, no viral vectors have been approved clinically for the treatment of CF, and this also applies to other diseases, particularly many other respiratory diseases.

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

[0008] Gene transfer efficiency to the airway epithelium is generally poor, at least in part because the receptors for many viral vectors appear to be located primarily on the basal surface of the airway epithelium. Therefore, prior to the inventors' research, using lentiviral pseudopattern specimens required disrupting epithelial integrity to transduce the airway, for example, through the use of cleansing agents such as lysophosphatidylcholine or ethylene glycol bis(2-aminoethyl ether)-N,N,N'N'-tetraacetic acid, which was associated with an increased risk of sepsis. Furthermore, conventional gene transfer vectors have difficulty penetrating the respiratory mucus layer, which also reduces gene transfer efficiency. The ability to repeatedly administer conventional viral vectors (essential for lifelong treatment of self-renewing epithelium) is limited by the patient's adaptive immune response, thus preventing successful repeated administration.

[0009] The delivery of the vector for clinical applications is another relevant factor. Therefore, it is essential to maintain viral stability achieved through the use of clinically relevant devices (such as bronchoscopes and nebulizers) to achieve therapeutic efficacy.

[0010] Therefore, there is a need for gene therapy vectors that can circumvent one or more of the problems described above. In particular, one object of the present invention is to provide a method for generating pseudo-patterned retroviral or lentiviral (e.g., SIV) vectors, and means for carrying out the method, wherein the resulting vectors are safe and suitable for improving trans-airway epithelial gene transfer efficiency and are produced on a clinically relevant scale. Summary of the Invention

[0011] The inventors previously developed a lentiviral vector that has been pseudomodeled from the hemagglutinin-neuraminidase (HN) and fusion (F) protein of a respiratory paramyxovirus, which contains a promoter and a transgenic gene. Typically, the vector backbone is derived from simian immunodeficiency virus (SIV), such as SIV1 or African green monkey SIV (SIV-AGM). Preferably, the backbone of the viral vector of the present invention is derived from SIV-AGM. The HN and F proteins are used to link to sialic acid and mediate cell fusion, respectively, so that the vector can enter target cells. The inventors have found that this specific F / HN pseudomodeled lentiviral vector can effectively transduce airway epithelium, resulting in the transgenic gene expression being maintained for a period exceeding the lifespan of the proposed airway epithelial cells. Importantly, the inventors have also found that re-administration does not lead to a loss of efficacy. These features make the carrier of the present invention an attractive candidate for treating diseases by means of its use in therapeutic proteins in the following ways: (i) within respiratory cells; (ii) secreted in the respiratory lumen; and (iii) secreted in the circulatory system.

[0012] However, this lentiviral vector presents potential safety concerns. In particular, it contains a large number of retroviral (i.e., non-transgenic) open reading frames (ORFs). The presence of these retroviral ORFs may pose a theoretical risk after administration to patients. The expression of retroviral ORFs implies a safety risk for patients, especially if the patient would have an immune response to the expressed retroviral sequence.

[0013] Furthermore, the significant sequence homology between the retroviral vector and the GagPol plasmids used in its production poses another theoretical risk: the potential generation of replication-competent lentiviruses (RCLs) during manufacturing or after administration to patients in clinical use. This represents another safety risk for patients. The risk of generating replication-competent viral particles is also a concern for other retroviral / lentiviral vectors.

[0014] While mitigating these risks would be desirable, it is not straightforward, or at least not without introducing other unacceptable drawbacks. On one hand, modifications that reduce the number of ORFs (especially the number of ORFs at the 5' of promoter transgenic genes) risk affecting the expression of downstream transgenic genes. Furthermore, other modifications to the retroviral genomic body (e.g., codon substitutions aimed at introducing stop codons to reduce retroviral ORF length) may also adversely affect, for example, vector yield and / or transgenic gene expression. Additionally, modifications known in this technology to reduce RCL risk (such as codon optimization for creating the gag-pol gene) typically have a negative impact on vector titer or yield. Given that high vector titers are required even for treating a single patient, such yield reductions could render its production commercially unfeasible.

[0015] As described herein, the inventors have designed and fabricated a retroviral vector, particularly a SIV vector, comprising a retroviral RNA sequence modified to reduce the number of retroviral ORFs and to introduce specific codon substitution modifications. The modified retroviral vectors of the present invention, comprising these newly described retroviral RNA sequences, mitigate one or more risks, providing a clinically advantageous product. Furthermore, the inventors have demonstrated that unexpected benefits can be obtained without anticipated disadvantages, such as reduced transgenic expression and / or decreased vector yield. While such modifications have been previously considered in the case of proviral DNA, this application is the first to elucidate such modifications within the retroviral / lentiviral RNA sequence itself, rather than within the manufacturing platform. Moreover, this application is the first to demonstrate the benefits derived from specific modifications to the retroviral / lentiviral RNA sequence, showing that this has a beneficial effect not only on vector yield but also on transgenic expression and the integration of the retroviral / lentiviral RNA sequence into host / target cells.

[0016] In particular, 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 at the 5' of the transgenic gene promoter, one or more SIV ORFs of a coding polypeptide of 100 amino acids or more, one or more ORFs contained (at least partially) in a portion of the RRE sequence, and / or contained (at least partially) in a portion of the RRE sequence. Gag One or more ORFs in the sequence. SIV ORF removal is achieved by removing the start codon (ATG) of the selected SIV ORF. To determine which SIV ORFs (and combinations thereof) can be removed without affecting the expression of downstream transgenic genes, the inventors produced a variety of different SIV vectors. Each SIV vector was evaluated to quantify vector yield and the expression of transgenic genes in modified SIV vectors versus their corresponding unmodified vectors.

[0017] The aforementioned modifications (codon substitution and reduction of the number of SIV ORFs) were confirmed to have no negative impact on the transgenic expression of SIV vectors pseudopatterned with hemagglutinin-neuraminidase (HN) and fusion (F) proteins derived from respiratory paramyxoviruses, and may even increase the transgenic expression of the vectors. This is unexpected, as it is generally believed that such modifications, while addressing potential safety concerns, may cause adverse effects on the transgenic expression.

[0018] Furthermore, the aforementioned mutations (both codon substitution and modification reducing the number of SIV ORFs) did not negatively impact the integration of SIV vectors pseudopatterned with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxoviruses into host / target cells, and may even have increased integration. This is also unexpected, as it is generally believed that such modifications, while addressing potential safety concerns, could adversely affect vector integration.

[0019] Furthermore, the aforementioned mutations (both codon substitution and modification reducing the number of SIV ORFs) did not negatively affect the yield of SIV vectors pseudopatterned by hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxoviruses, and even increased vector titer. This was also unexpected, as it was generally believed that such modifications, while addressing potential safety issues, could adversely affect vector yield.

[0020] Therefore, the present invention provides a retroviral vector comprising a modified retroviral RNA sequence, wherein the modified retroviral RNA sequence is (i) codon-substituted and (ii) contains a reduced number of retroviral open reading frames (ORFs) compared to an unmodified retroviral RNA sequence from which the modified retroviral RNA sequence is derived; and wherein: (a) the retroviral RNA sequence contains a promoter and a transgenic gene; and (b) the retroviral vector is pseudo-patterned with hemagglutinin-neuronalase (HN) and fusion (F) proteins derived from respiratory paramyxovirus.

[0021] Also disclosed is a method for generating retroviral (especially lentiviral) vectors (such as SIVs) containing a retroviral RNA sequence that is codon-substituted and contains a reduced number of retroviral OFRs compared to an unmodified plasmosome vector from which the modified retroviral genome RNA sequence is derived, wherein (a) the retroviral RNA sequence contains a promoter and a transgenic gene, and (b) the retroviral vector is pseudopatterned with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxoviruses, wherein the retroviral vector has a reduced risk of immune response when administered to a patient and does not negatively affect the expression of the transgenic gene.

[0022] The modified retroviral genome RNA sequence may lack: (a) one or more retroviral ORFs at the 5' of the promoter; (b) one or more retroviral ORFs encoding a polypeptide of ≥100 amino acids in length; (c) a sequence contained (at least partially) in part of the promoter. RREOne or more retroviral ORFs in the sequence; and / or (d) contained (at least partially) in a portion Gag One or more retroviral ORFs in the sequence.

[0023] This respiratory paramyxovirus could be Sendai virus.

[0024] The promoter can be selected from the following groups: heterozygous human CMV enhancer / EF1a (hCEF) promoter, cytomegalovirus (CMV) promoter, and elongation factor 1a (EF1a) promoter. Preferably, the vector may contain the heterozygous human CMV enhancer / EF1a (hCEF) promoter.

[0025] The transgenic gene may be selected from: (a) CFTR, ABCA3, DNAH5, DNAH11, DNAI1, and DNAI2; or (b) a secreted therapeutic protein, optionally α-1 antitrypsin (A1AT), factor VIII, surfactant protein B (SFTPB), factor VII, factor IX, factor X, factor XI, von Willebrand factor, granulocyte-macrophage community-stimulating factor (GM-CSF), and monoclonal antibodies against the vector. Preferably, the transgenic gene may encode: (a) CFTR; (b) A1AT; or (c) FVIII.

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

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

[0028] The modified retroviral RNA sequence may (i) be less than 9,000 bases in length; and / or (ii) contain 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 with SEQ ID NO: 1. Preferably, the modified retroviral RNA sequence may (i) be less than 9,000 bases in length; and (ii) contain 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 with SEQ ID NO: 1. More preferably, the modified retroviral RNA sequence may contain 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 may further comprise one or more of the following: (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 with 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 with SEQ ID NO: 3; and (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 with SEQ ID NO: 2. 4. 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 with SEQ ID NO: 5, or composed of such amino acid sequence; (d) A protease comprising or composed 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 with SEQ ID NO: 5; (e) A p51 protein comprising or composed 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 with SEQ ID NO: 6; (f) A p15 protein comprising or composed 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 with SEQ ID NO: 5. 7. An amino acid sequence having or consisting of at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity with SEQ ID NO: 8; and / or (g) a p31 protein comprising or consisting of an amino acid sequence having or consisting of at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity with 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 the following: (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 with 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 with SEQ ID NO: 10.

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

[0032] Also disclosed is a method for producing retroviral (especially lentiviral) vectors (such as SIVs) comprising a retroviral RNA sequence that is codon-substituted and contains a reduced number of retroviral OFRs compared to an unmodified plasmosomal vector from which the modified retroviral genome RNA sequence is derived, wherein (a) the retroviral RNA sequence contains a promoter and a transgenic gene, and (b) the retroviral vector is pseudopatterned with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxoviruses, wherein the method reduces the risk of recurrent viral load (RCL) and does not negatively affect or even improve vector titer, vector integration, and / or transgenic gene expression. Therefore, the method of the present invention provides a safer vector produced in commercially desirable quantities.

[0033] Therefore, the present invention also provides a method for producing a retroviral vector, wherein the retroviral vector is codon-substituted and contains a reduced number of ORFs compared to an unmodified retroviral RNA sequence from which a modified retroviral RNA sequence is derived, wherein the retroviral RNA sequence contains a promoter and a transfection gene, and wherein the retroviral vector is pseudo-patterned with hemagglutinin-neuraminidase (HN) and fusion (F) proteins derived from respiratory paramyxovirus. The method of the present invention may comprise or consist of the following steps: (a) suspension growth of cells; (b) transfection of these cells with one or more plastids; (c) addition of nucleases; (d) collection of lentiviruses; (e) addition of trypsin (or an enzyme with the same cleavage specificity); and (d) purification.

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

[0035] The present invention further provides a retroviral vector, which, compared with an unmodified retroviral RNA sequence from which a modified retroviral RNA sequence is derived, has undergone codon substitution and contains a reduced number of ORFs, wherein the retroviral RNA sequence contains a promoter and a transgenic gene, and the retroviral vector is pseudo-patterned by hemagglutinin-neuronalase (HN) and fusion (F) protein from respiratory paramyxovirus, and the retroviral vector 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, the modified retroviral RNA sequence being codon-substituted and containing a reduced number of ORFs compared to an unmodified retroviral RNA sequence from which the modified retroviral RNA sequence is derived, and wherein the retroviral RNA sequence contains a promoter and a transgenic gene, and the retroviral vector is pseudo-patterned with hemagglutinin-neuraminidase (HN) and fusion (F) proteins derived from respiratory paramyxoviruses. The composition can be formulated for pulmonary administration; optionally, the administration can be performed via intratracheal or intranasal instillation, aerosol delivery, intravenous injection, or direct pulmonary injection.

[0037] The present invention also provides a retroviral vector for use in a treatment method, wherein the retroviral vector comprises a modified retroviral RNA sequence, the modified retroviral RNA sequence having codon substitutions and containing a reduced number of ORFs compared to an unmodified retroviral RNA sequence from which the modified retroviral RNA sequence is derived, and wherein the retroviral RNA sequence comprises a promoter and a transgenic gene, and the retroviral vector is pseudo-patterned with hemagglutinin-neuronalglucosidase (HN) and fusion (F) proteins from respiratory paramyxovirus. The present invention also provides a method for treating a disease, comprising administering a retroviral vector to a subject in need, wherein the retroviral vector comprises a modified retroviral RNA sequence, the modified retroviral RNA sequence being codon-substituted and containing a reduced number of ORFs compared to an unmodified retroviral RNA sequence from which the modified retroviral RNA sequence is derived, and wherein the retroviral RNA sequence comprises a promoter and a transgenic gene, and the retroviral vector is pseudopatterned with hemagglutinin-neuraminidase (HN) and fusion (F) proteins derived from respiratory paramyxovirus. The disease to be treated may be a lung disease, preferably cystic fibrosis. Attached Figure Description

[0038] Figures 1A to 1F A schematic diagram showing an example of the properties of the carrier used to generate the present invention. Figure 1G Displaying unmodified vector gene somatoforms.

[0039] Figure 2 Demonstrating the means for generating the present invention A1AT A schematic diagram of an exemplary pDNA1 plasmid of the vector.

[0040] Figures 3A to 3D Demonstrating the F used to generate the present invention VIII A schematic diagram of an exemplary pDNA1 plasmid of the vector.

[0041] Figure 4 The diagram shows fourteen ATG start codons present in the Gag-RRE region of the pGM326 gene somatoform, which can produce ORFs longer than 10 amino acids. Arrows depict ORFs that can be generated by each of the labeled start codons. Circled ATGs are ATGs with strong kozak and are framed alongside Gag or Env.

[0042] Figure 5 The SIV-CFTR titer (TU / mL) of LV produced using the Ambr® 15 bioreactor system is shown, as evaluated by A549 FACS analysis. VRC = Vector reference control.

[0043] Figure 6 The SIV-CFTR titer (TU / mL) of LV produced using the Ambr® 15 bioreactor system is shown, assessed by HEK293T day 3 integration assay. Clear bars indicate values ​​below the limit of quantitation. VRC = vector reference control. Cell-extracted DNA collected at day 3 was size-selectively purified to remove unintegrated DNA and analyzed by qPCR.

[0044] Figure 7 A549 cells expressing the CFTR protein are shown as a percentage of the live single-cell population as analyzed by FACS. VRC = vector reference control; sample diluted 1:20.

[0045] Figure 8 The Western ink dot method (using anti-PIV1 antibody ab20791 diluted 1:5000) shows that Fct4 is cleaved by the trypsin-like enzyme TrypLE. Detailed Implementation

[0046] 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 invention pertains. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20th edition, John Wiley and Sons, New York (1994), and Hale and Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide general dictionaries for those skilled in the art regarding many of the terms used in this invention. The meanings and scopes of these terms are clear; however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definition. It should be understood that this invention is not limited to the specific methodologies, schemes, and reagents described herein and is therefore subject to variation.

[0047] This invention is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar to or equivalent to those described herein may be used to practice or test embodiments of the invention. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined solely by the claims.

[0048] The description of embodiments of the present invention is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Those skilled in the art will recognize that while specific embodiments and examples of the invention have been described herein for illustrative purposes, various equivalent modifications are possible within the scope of the invention. For example, although method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order or substantially simultaneously. The teachings of the invention provided herein can be applied to other procedures or methods as needed. The various embodiments described herein can be combined to provide other embodiments. Where necessary, the content of the invention can be modified to incorporate the compositions, functions, and concepts of the foregoing references and applications, thereby providing other embodiments of the invention. Furthermore, due to biological functional equivalence considerations, some changes can be made to the protein structure without affecting the type or amount of biological or chemical action. Such and other changes can be made to the invention according to the implementation. All such modifications are intended to be included within the scope of the appended claims.

[0049] Unless otherwise instructed, any nucleic acid sequence shall be written from left to right with a 5' to 3' orientation; amino acid sequences shall be written from left to right with an amino group to a carboxyl group orientation.

[0050] The titles provided herein are not intended to limit the scope or embodiments of the invention.

[0051] As used in this article, the term “capable” when used with a verb encompasses or implies the action of the corresponding verb. For example, “capable of interacting” also means interacting, “capable of splitting” also means splitting, “capable of combining” also means combining, and “capable of specifically targeting” also means specifically targeting.

[0052] Other definitions of the terminology may be presented throughout the specification. Before describing the exemplary embodiments in more detail, it should be understood that the invention is not limited to the specific embodiments described and is therefore subject to variation. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of the invention will be defined only by the appended claims.

[0053] Numerical ranges include the numerical values ​​that define the range. Where a range of values ​​is provided, it should be understood that, unless the context explicitly specifies otherwise, each interpolated value between the upper and lower limits of the range is also specifically disclosed, accurate to the tenths of the lower limit unit. This invention covers all smaller ranges between any stated or interpolated values ​​within the stated range, and any other stated or interpolated value within the stated range. The upper and lower limits of these smaller ranges may be independently included or excluded from the range, and any limit value, infinity, or both limits included in the smaller range are also covered by this invention, but are subject to any specifically excluded limit value within the stated range. Where a stated range includes one or both of the limit values, this invention also includes ranges that exclude either or both of those included limit values.

[0054] As used herein, the article “a (and an)” may refer to one or more (e.g., at least one) grammatical objects. Furthermore, unless the context otherwise requires, singular terms should include plurals and plural terms should include singulars. In this application, unless otherwise stated, the use of “or” means “and / or”. Furthermore, the use of the term “including” and other forms such as “includes” and “included” is not restrictive.

[0055] The term "about" generally refers to the acceptable degree of error in a measured quantity given the nature or precision of the measurement. Exemplary error levels are within 20 percent (%) of a given value or range of values, typically within 10%, and more usually within 5%. Preferably, the term "about" as used herein is understood to mean the numerical value used with it plus or minus (±) 5%, more preferably ±4%, ±3%, ±2%, ±1%, ±0.5%, or ±0.1%.

[0056] The term “composed of” refers to compositions, methods and their components as described herein, excluding any elements not listed in this description of the invention.

[0057] As used herein, the term "consistently of" refers to those elements required for a given invention. This term allows for the presence of elements (i.e., inactive or non-immunogenic components) that do not materially affect the basic, novel, or functional features of the invention.

[0058] The embodiments described herein as “comprising” one or more features may also be regarded as disclosures of corresponding embodiments that are “consistent with such features” and / or “substantially constitute with such features”.

[0059] Concentration, amount, volume, percentage and other values ​​may be presented in range form in this document. It should also be understood that such range form is used for convenience and brevity purposes only, and should be interpreted flexibly to include not only the values ​​explicitly listed in the range limits, but also all individual values ​​or subranges covered in the range, as if each value and subrange were explicitly listed.

[0060] As used herein, unless otherwise stated, the terms “vector,” “retroviral vector,” and “retroviral F / HN vector” are used interchangeably to mean a retroviral vector containing a retroviral RNA sequence and pseudopatterned with hemagglutinin-neuroglucosidase (HN) and fusion (F) proteins from respiratory paramyxoviruses. Unless otherwise stated, the terms “lentiviral vector” and “lentiviral F / HN vector” are used interchangeably to mean a lentiviral vector pseudopatterned with hemagglutinin-neuroglucosidase (HN) and fusion (F) proteins from respiratory paramyxoviruses. All disclosures herein regarding the retroviral vectors of the present invention are equally and unreservedly applied to the lentiviral vectors of the present invention, and to SIV vectors pseudopatterned with hemagglutinin-neuroglucosidase (HN) and fusion (F) proteins from respiratory paramyxoviruses (also referred to herein as SIV F / HN or SIV-FHN).

[0061] As defined herein, the term "retroviral RNA sequence" refers to a nucleic acid molecule contained within a retroviral vector. A retroviral RNA sequence comprises a long terminal repeat (LTR) component, the nucleic acid sequence required to incorporate the retroviral RNA sequence into the retroviral particle, and a transgenic expression cartridge. The transgenic expression cartridge contains suitable enhancer / promoter components, transgenic cDNA, and post-transcriptional regulatory components. Retroviral RNA sequences typically begin with a 5' LTR R sequence and typically end with a 3' LTR R sequence. The 5' region of the retroviral RNA sequence typically contains a retroviral LTR R sequence followed by a retroviral LTR U5 sequence (in 5' to 3' order), or consists of these sequences. The 3' region of the retroviral RNA sequence typically contains a retroviral LTR U3 sequence followed by a retroviral LTR R sequence (in 5' to 3' order), or consists of these sequences.

[0062] The terms "DNA provirus" or "DNA provirus sequence" and "DNA proviral sequence" are used interchangeably to refer to the DNA sequence integrated into the genome of a retroviral transduced cell. DNA provirus sequences contain additional nucleic acid regions not found in retroviral RNA sequences, including the 5' LTR U3 and 3' LTR U5 sequences. Therefore, the DNA provirus sequence and the retroviral RNA sequence are not identical; rather, the retroviral RNA sequence is shorter than the proviral DNA sequence from which it is derived. The precise 5' and 3' boundaries of the retroviral RNA sequence cannot be easily and reliably determined by simple analysis of the proviral DNA sequence compared to the proviral DNA sequence from which the retroviral RNA sequence is derived.

[0063] The retroviral vector of the present invention comprises a codon-substituted retroviral RNA sequence. Those skilled in the art will understand 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 thus reducing the length of any encoded retroviral (poly)peptide, and / or increasing the translation efficiency of the coding gene. Translation efficiency can be improved by modifying the nucleic acid sequence. Codon substitution is conventional in this art, and designing codon-substituted patterns for a given nucleic acid sequence is within the scope of conventional practice for those skilled in the art. However, predicting the effects of codon substitution on other parameters is not straightforward. As a non-limiting example, as described herein, prior knowledge teaches that under normal manufacturing conditions, codon substitution can reduce vector yield and / or transfected gene expression.

[0064] In addition to codon substitution, the retroviral RNA sequence of this invention further includes modifications to reduce the number of retroviral open reading frames (ORFs). Those skilled in the art will understand that an open reading frame is a DNA or RNA sequence between a start codon and a stop codon. ORFs can be easily identified from the NIH using standard techniques known in the art, such as software tools like ORFfinder (ORFfinder Home - NCBI (nih.gov)). Standard methods for testing the effects of ORFs on, for example, vector yield and / or transgenic gene expression are also within the ordinary skill of those skilled in the art, and exemplary methods are described herein. A retroviral ORF is an ORF present in a (unmodified) retroviral RNA sequence that can potentially be expressed in a patient to produce retroviral proteins. Partially or completely overlapping ORFs typically occur on the same nucleic acid strand. Furthermore, competing ORFs typically occur on different nucleic acid strands. Upon administration of a retroviral vector, expression of one or more retroviral open reading frames (ORFs) to produce retroviral proteins can theoretically trigger an immune response. Specifically, in this context, the terms "ORF reduction," "ORF elimination," and "ORF disruption" can be used interchangeably to refer to the removal of open reading frames, i.e., reducing the number of ORFs translated to represent retroviral protein, peptide, or polypeptide sequences. This can be achieved by any suitable technique, such as by deleting the start codon (also known as the initiation codon) of the ORF. Alternatively, the nucleotides in the start codon can be substituted, or one or more additional nucleotides can be added to disrupt the start codon. Those skilled in this art will further understand that the start codon in the retroviral RNA sequence is AUG, and the corresponding start codon in the proviral DNA sequence is ATG.

[0065] The stop codon signals the termination of translation. Those familiar with this technique should know that the standard stop codons in retroviral RNA sequences can be selected from UAG, UAA, and UGA. The corresponding standard stop codons in proviral DNA sequences are TAG, TAA, and TGA.

[0066] The retroviral vector of the present invention may additionally comprise a codon-optimized retroviral RNA sequence. Those skilled in the art will understand that codon optimization is a technique for maximizing protein expression. For example, codon optimization can improve the translation efficiency of coding genes. Translation efficiency can be improved by modifying nucleic acid sequences. Codon optimization is routine in this art, and designing codon-optimized forms of a given nucleic acid sequence is within the scope of conventional practice for those skilled in the art. However, predicting the effects of codon optimization on other parameters is not straightforward. As a non-limiting example, as described herein, prior knowledge teaches that under normal manufacturing conditions, codon optimization of the gag-pol gene typically reduces vector yield.

[0067] As used herein, the terms “potency” and “yield” are used interchangeably, referring to the amount of lentiviral (e.g., SIV) vector produced by the method of this invention. Potency is a primary benchmark for characterizing manufacturing efficiency, with higher potency generally indicating the production of more retroviral / lentiviral (e.g., SIV) vector (e.g., using the same amount of reagent). Potency or yield can be correlated with the number of vector genomes integrated into the target cell genome (integration potency), which is a measure of “active” viral particles, i.e., the number of particles capable of transducing cells. Transduction unit (TU / mL, also known as TTU / mL) is a biological reading of the number of host cells transduced under certain tissue culture / virus dilution conditions and is a measure of the number of “active” viral particles. The total number of (active + inactive) viral particles can also be determined by any suitable means, such as by measuring how much Gag or viral RNA copy is present in the test solution. It is then assumed that the lentiviral particle contains 2000 Gag molecules or 2 viral RNA molecules. Once the total number of particles and transduction titer / TU have been measured, the particle:infectivity ratio can be calculated. Amino acids are referred to in this article by their names, three-letter abbreviations, or single-letter abbreviations.

[0068] As used herein, the terms “protein” and “peptide” are used interchangeably to refer to a series of amino acid residues linked together by peptide bonds between the α-amine and carboxyl groups of adjacent residues. The terms “protein” and “peptide” refer to amino acids, including polymers of modified amino acids (e.g., phosphorylated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. “Protein” and “peptide” are generally used when referring to relatively large peptides, while the term “peptide” is generally used when referring to smaller peptides; however, in this art, the use of these terms overlaps. When referring to gene products and their fragments, the terms “protein” and “peptide” are used interchangeably herein. Therefore, exemplary peptides or proteins include gene products, naturally occurring proteins, homologs, heterohomologs, homohomologs, fragments, and other equivalents, variants, fragments, and analogs thereof.

[0069] As used herein, the terms “polynucleotide,” “nucleic acid,” and “nucleic acid sequence” refer to any molecule, preferably a polymer, incorporating units of ribonucleic acid, deoxyribonucleic acid, or similar molecules. Nucleic acids can be single-stranded or double-stranded. A single-stranded nucleic acid can be a single strand of denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including siRNA, shRNA, and antisense oligonucleotides. The terms “transgenic gene” and “gene” are used interchangeably, and both terms encompass the segment or variant encoding the target protein.

[0070] The transgenic genes of the present invention include nucleic acid sequences removed from their natural environment, recombinant or selected DNA isolates, and chemically synthesized analogs or analogs biosynthesized through heterologous systems.

[0071] Minor variations in the amino acid sequence of this invention are covered by this invention, provided that the variations 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 sequence of this invention or any fragment thereof as defined anywhere herein. The term homology is used herein to mean identity. Therefore, the sequences of variants or analogues of the amino acid sequence of this invention may differ based on substitution (usually conserved substitution), deletion, or insertion. Proteins containing such variations are referred to herein as variants.

[0072] The proteins of this invention may include variants in which amino acid residues from one species are substituted at conserved or non-conserved positions with corresponding residues from another species. Variants of the protein molecules disclosed herein can be generated and used in this invention. Following the guidance of computational chemistry in applying multivariate data analysis techniques to structure / property-activity relationships [see, for example, 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, graph recognition, and classification [see, for example, 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, Vol. 22 (Paper Edition)). Oxford University Press; (December 2000), ISBN: 0198507089; Witten, Ian H. et al. Data Mining: Practical Machine Learning Tools and Techniques with Java Implementations. Morgan Kaufmann; (October 11, 1999), ISBN: 1558605525; Denison David GT (ed.) 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 possible contact residues or calculated physicochemical properties), and these properties can be considered individually and in combination.

[0073] Amino acids are referred to herein by their names, three-letter abbreviations, or single-letter abbreviations. As used herein, the term "protein" includes proteins, polypeptides, and peptides. As used herein, the term "amino acid sequence" is synonymous with the terms "polypeptide" and / or "protein." In some cases, the term "amino acid sequence" is synonymous with the term "peptide." The terms "protein" and "polypeptide" are used interchangeably herein. Common single-letter and three-letter codes for amino acid residues may be used within the scope of this invention and the claims. Three-letter codes for amino acids are used if they conform to the definition of the Joint Commission on Biochemical Nomenclature (JCBN) of IUPACIUB. It should also be understood that, due to the degeneracy of the genetic code, polypeptides can be encoded by more than one nucleotide sequence.

[0074] Amino acid residues at non-conserved positions can be substituted by either conserved or non-conserved residues. In particular, conserved amino acid substitutions should be considered.

[0075] "Conservative amino acid substitution" refers to the substitution of an amino acid residue by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in this technique, including basic side chains (e.g., lysine, arginine, or histidine), acidic side chains (e.g., aspartic acid or glutamic acid), nonpolar side chains (e.g., glycine, asparagine, glutamic acid, serine, threonine, tyrosine, or cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, tryptophan, or histidine). Therefore, if an amino acid in a polypeptide is substituted by another amino acid from the same side chain family, the amino acid substitution is considered conservative. The proteins of this invention include conserved variants, but do not exclude other forms of variants, such as polymorphic variants, interspecific homologs, and paired genes.

[0076] “Non-conservative amino acid substitution” includes the following, wherein (i) a residue with an electronegative side chain (e.g., Arg, His, or Lys) substitutes for or is substituted by an electronegative residue (e.g., Glu or Asp); (ii) a hydrophilic residue (e.g., Ser or Thr) substitutes for or is substituted by a hydrophobic residue (e.g., Ala, Leu, Ile, Phe, or Val); (iii) a cysteine ​​or proline substitutes for or is substituted by any other residue; or (iv) a residue with a large hydrophobic or aromatic side chain (e.g., Val, His, Ile, or Trp) substitutes for a residue with a smaller side chain (e.g., Ala or Ser) or a residue without a side chain (e.g., Gly) or is substituted by or is substituted by a residue with a smaller side chain (e.g., Ala or Ser) or a residue without a side chain (e.g., Gly).

[0077] "Insertion" or "deletion" typically occurs within the range of about 1, 2, or 3 amino acids. Permissible variations can be determined experimentally by systematically introducing amino acid insertions or deletions into proteins using recombinant DNA technology and analyzing the activity of the resulting recombinant variants. For those familiar with this technique, this requires only routine experiments.

[0078] A “fragment” of a polypeptide contains 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.

[0079] The polynucleotides of this invention can be prepared by any means known in this art. For example, large quantities of polynucleotides can be produced by replication in a suitable host cell. The natural or synthetic DNA fragment encoding the desired segment is incorporated into a recombinant nucleic acid construct, typically a DNA construct, capable of being introduced into and replicated in prokaryotic or eukaryotic cells. Typically, the DNA construct will be adapted for autonomous replication in a single-celled host (such as yeast or bacteria), but may also be intended for introduction into and integration into the genome of cultured insects, mammals, plants, or other eukaryotic cell lines.

[0080] The polynucleotides of the present invention can also be produced by chemical synthesis, for example by the ammonium phosphite method or the triester method, and can be carried out on a commercially available automated oligonucleotide synthesizer. Double-stranded fragments can be obtained from chemically synthesized single-stranded products by synthesizing complementary strands and bonding them together under appropriate conditions, or by adding complementary strands using DNA polymerase with appropriate primer sequences.

[0081] When applied to nucleic acid sequences, in the context of this invention, the term "isolated" means that the polynucleotide sequence has been removed from its native genetic environment and therefore does not contain other foreign or unwanted coding sequences (but may include naturally present 5' and 3' untranslated regions, such as promoters and terminators), and is in a form suitable for use in a genetically engineered protein production system. Such isolated molecules are molecules isolated from their native environment.

[0082] Given the degeneracy of the genetic code, considerable sequence variation may exist in the polynucleotides of this invention. The degenerate codons covering all possible codons for a given amino acid are described below: amino acids codon Degenerate codons Cys TGC TGT TGY Ser AGC AGT TCA TCC TCG TCT WSN Thr ACA ACC ACG ACT ACN Pro CCA CCC CCG CCT CCN Ala GCA GCC GCG GCT GCN Gly GGA GGC GGG GGT GGN Asn AAC AAT AAY Asp GAC GAT GAY Glu GAA GAG GAR Gln CAA CAG CAR His CAC CAT CAY Arg AGA AGG CGA CGC CGG CGT MGN Lys AAA AAG AAR Met ATG ATG Ile ATA ATC ATT ATH Leu CTA CTC CTG CTT TTA TTG YTN Val GTA GTC GTG GTT GTN Phe TTC TTT TTY Tyr TAC TAT TAY Trp TGG TGG Ter TAA TAG TGA TRR Asn / Asp RAY Glu / Gln SAR Any NNN Those skilled in the art will understand that there is flexibility in determining degenerate codons, which represent all possible codons encoding each amino acid. For example, some polynucleotides covered by degenerate sequences may encode variant amino acid sequences, but those skilled in the art can easily identify such variant sequences by referring to the amino acid sequences of this invention.

[0083] The “mutated” nucleic acid sequence is substantially homologous or substantially similar to a reference nucleic acid sequence (or a fragment thereof). A nucleic acid sequence or fragment thereof is considered “substantially homologous” (or “substantially identical”) to a reference sequence if, upon optimal alignment with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, nucleotide sequence identity is found in at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or a higher percentage of nucleotide bases. Methods for determining the homology of nucleic acid sequences are known in this art.

[0084] Alternatively, if the "variant" and the reference sequence can hybridize under strict (e.g., highly strict) heterozygous conditions, the "variant" nucleic acid sequence is substantially homologous (or substantially identical) to the reference sequence (or fragments thereof). As will be readily apparent to those familiar with this technique, in addition to base composition, complementary strand length, and the number of nucleotide base mismatches between the hybrid nucleic acids, nucleic acid sequence heterozygosity is also affected by conditions such as salt concentration (e.g., NaCl), temperature, or organic solvents. Strict temperature conditions are preferred, and generally include temperatures above 30°C, typically above 37°C, and preferably above 45°C. Strict salt conditions are typically 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 far more important than any single parameter.

[0085] Methods for determining the percentage of nucleic acid sequence identity are known in this art. For example, in assessing nucleic acid sequence identity, a sequence having a defined number of consecutive nucleotides can be compared with a corresponding portion of a nucleic acid sequence from the present invention (having the same number of consecutive nucleotides). Tools known in this art for determining the percentage of nucleic acid sequence identity include nucleotide BLAST (described below).

[0086] Those familiar with this technique will understand that different species exhibit "preferred codon usage." As used herein, the term "preferred codon usage" refers to the most frequently used codon in the cells of a particular species, thus favoring one or more representatives 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 frequently used codon; in other species, different codons may be preferred. Preferred codons of a specific host cell species can be introduced into the polynucleotides of the present invention using various methods known in this technique. Introducing preferred codon sequences into recombinant DNA can, for example, enhance protein production by making protein translation more efficient in a specific cell type or species. Therefore, according to the present invention, any nucleic acid sequence other than the gag-pol gene can be codon-optimized for expression in host or target cells. In particular, the vector gene (or its corresponding plastid), the REV gene (or its corresponding plastid), the fusion protein (F) gene (or its corresponding plastid) and / or the hemagglutinin-neuraminidase (HN) gene (or its corresponding plastid), or any combination thereof, may be codon-optimized.

[0087] A “fragment” of the polynucleotide of interest comprises a series of consecutive nucleotides from the sequence of the full-length polynucleotide. For example, a “fragment” of the polynucleotide of interest may comprise or consist of at least 30 consecutive nucleotides from the sequence of the 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 consecutive nucleic acid residues of the polynucleotide). The fragment may include at least one antigenic determinant corresponding to the polypeptide of interest and / or may encode at least one antigenic epitope corresponding to the polypeptide of interest. Generally, fragments as defined herein retain the same function as the full-length polynucleotide.

[0088] The terms “decrease,” “reduced / reduction,” or “inhibit” are used herein to mean a statistically significant reduction. The terms “decrease,” “reduction,” or “inhibit” generally mean a reduction of at least 10% compared to a reference level (e.g., without a given treatment), and may 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, “decrease” or “inhibit” encompasses complete inhibition or reduction compared to a reference level. “Complete inhibition” means 100% inhibition (i.e., elimination) compared to a reference level.

[0089] The terms “increased / increase,” “enhance,” or “activate” are used herein to mean an increase in a statistically significant amount. “Increased,” “enhance,” or “activate” may mean an increase of at least 25%, at least 50%, for example, an increase of 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 the reference level, or an increase of at least about 1.5 times, or at least about 2 times, or at least about 2.5 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times compared to the reference level, or any increase between 1.5 times and 10 times or greater. In the case of yield or potency, “increase” means an observable or statistically significant increase in such levels.

[0090] The terms “individual,” “subject,” and “patient” are used interchangeably herein to refer to a mammalian subject requiring diagnosis, prognosis, disease monitoring, treatment, therapy, and / or therapy optimization. Mammals may be (but are not limited to) humans, non-human primates, mice, rats, dogs, cats, horses, or cattle. In a preferred embodiment, the individual, subject, or patient is a human. An “individual” may be an adult, adolescent, or infant. An “individual” may be male or female.

[0091] The “object” who “needs” treatment for a specific condition can be an individual who has the condition, has been diagnosed with the condition, or is at risk of developing the condition.

[0092] The subject may be a person who has been previously diagnosed with, identified as having, or has a condition requiring treatment, or one or more complications or symptoms related to such a condition, and optionally has received treatment for the condition or one or more complications or symptoms related to such condition as defined herein. Alternatively, the subject may be a person who has not previously been diagnosed with a condition as defined herein, or one or more symptoms or complications related to such condition. For example, the subject may be a person exhibiting the condition or one or more risk factors for one or more symptoms or complications related to such condition, or a person who does not exhibit risk factors.

[0093] As used herein, the term "healthy individual" refers to an individual or group of individuals in a healthy state, such as an individual who has not yet exhibited any symptoms of disease, has not been diagnosed with a disease, and / or is unlikely to have a disease (e.g., cystic fibrosis (CF) or any other disease described herein). Preferably, the healthy individual is not taking any medications that affect CF and has not been diagnosed with any other disease. Compared to the tested individuals, one or more healthy individuals may have similar sex, age, and / or body mass index (BMI). Standard statistical methods used in applied medicine allow for the determination of normal performance levels in healthy individuals and significant deviations from such normal levels.

[0094] In this article, the terms “control group” and “reference group” are used interchangeably.

[0095] As used in this article, the term "medically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia, the European Pharmacopeia, or other recognized pharmacopoeias.

[0096] The disclosures discussed herein are provided only for information prior to the filing date of this application. Nothing in this document should be construed as an admission that such disclosures constitute prior art to the claims covered herein.

[0097] The disclosures relating to the various methods of the present invention are intended to be equally applicable to other methods, therapeutic uses or approaches, data storage media or devices, computer program products, and vice versa.

[0098] Retroviruses and Lentiviral Vectors This invention relates to a retroviral / lentiviral (e.g., SIV) vector. The term "retrovirus" refers to any member of the RNA virus family Retroviridae encoding the enzyme reverse transcriptase. The term "lentivirus" refers to a retroviridae family. Examples of retroviruses suitable for this invention include gamma retroviruses, such as murine leukemia virus (MLV) and feline leukemia virus (FLV). Examples of lentiviruses suitable for this invention include simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), and Bisnay / Medy virus. Preferably, this invention relates to lentiviral vectors and their generation. Particularly preferred lentiviral vectors are SIV vectors (including all viral strains and subtypes), such as SIV-AGM (originally derived from African green savanna monkeys). Cercopithecus aethiops (Separation). Alternatively, this invention relates to HIV vectors.

[0099] The retroviral / lentiviral (e.g., SIV) vectors of the present invention are typically pseudo-patterned using hemagglutinin-neuraminidase (HN) and fusion (F) proteins derived from respiratory paramyxoviruses. Preferably, the respiratory paramyxovirus is Sendai virus (type 1 rodent parainfluenza virus).

[0100] The F protein may be a truncated F protein, typically a protein in which the cytoplasmic domain has been truncated. Preferably, the truncated F protein is Fct4, ​​in which 38 amino acids have been removed from the C-terminus of the F protein, retaining 4 amino acids of the cytoplasmic domain of the F protein. Therefore, the F protein may contain or consist of an Fct4 amino acid sequence that is 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 higher, and at most 100% identical to SEQ ID NO: 12 or 13. Preferably, the F protein may contain or consist of an Fct4 amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 12 or 13.

[0101] Full-length F proteins or their C-terminal truncated forms (such as Fct4) are typically fusion-inactive. The fusion-inactive form of the F protein can be cleaved to produce two subunits: the first unit (also known as F2) and the second unit (also known as F1).

[0102] The first unit 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 higher, and at most 100% identical to, SEQ ID NO: 14. Preferably, the first unit may be a second unit comprising or consisting of an amino acid sequence having at least 90%, at least 95%, or at least 99% identical to, SEQ ID NO: 14. SEQ ID NO: 14 is the first unit of Fct4.

[0103] Alternatively, and preferably additionally, the second unit 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 higher, and at most 100% identical to, SEQ ID NO: 15. Preferably, the second unit may be a subunit comprising or consisting of an amino acid sequence having at least 90%, at least 95%, or at least 99% identical to, SEQ ID NO: 15. SEQ ID NO: 15 is the second unit of Fct4.

[0104] F proteins (e.g., Fct4) may contain an N-terminal signal peptide. Alternatively, F proteins may lack such a signal peptide. The F protein signal peptide may contain or consist of an amino acid sequence that is 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 higher, and at most 100% identical to, SEQ ID NO: 16. This signal peptide can 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. Therefore, the mature form of Fct4 may contain or consist of an amino acid sequence that is 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 higher, and at most 100% identical to or composed of the amino acid sequence of amino acid residues 26-527 of SEQ ID NO: 13.

[0105] Within the exemplary F protein body (pDNA3a) pGM301, a potential alternative start codon exists upstream of the start codon that initiates translation to produce Fct4 in SEQ ID NO: 12 and 13. However, according to the present invention, the F protein of the retroviral / lentiviral (e.g., SIV) vector of the present invention does not contain an additional amino acid sequence at the N-terminus of methionine at position 1 in SEQ ID NO: 13. In particular, the F protein of the retroviral / lentiviral (e.g., SIV) vector of the present invention generally does not contain one or more amino acids corresponding to those encoded by bases 1645-1734 of pGM301 (SEQ ID NO: 23), which, after translation, becomes MFMPSSFSYSSWATCWLLCCLIILAKNSIA (SEQ ID NO: 46) at the N-terminus of methionine at position 1 in SEQ ID NO: 13.

[0106] HN proteins can be truncated and / or chimeric HN proteins, typically HN proteins in which the cytoplasmic domain has been truncated or replaced. Preferably, the HN protein is a chimeric HN protein in which (i) the cytoplasmic domain of the HN is replaced by the cytoplasmic domain of a transmembrane (TMP) protein; or (ii) the cytoplasmic domain of the TMP is added to the cytoplasmic domain of the HN protein. HN proteins can be as described by Kobayashi et al. (J. Virol. (2003) 77(4): 2607-2614), which is incorporated herein by reference in its entirety.

[0107] F / HN pseudomodeling is particularly effective in targeting cells in the airway epithelium, and therefore, for therapeutic applications, it is typically delivered to cells of the respiratory tract, including airway epithelial cells. Therefore, the retroviral / lentiviral (e.g., SIV) vector of the present invention is particularly suitable for treating diseases or conditions of the airways, respiratory tract, or lungs. Typically, retroviral / lentiviral (e.g., SIV) vectors can be used to treat hereditary respiratory diseases.

[0108] The retroviral / lentiviral (e.g., SIV) vectors of the present invention can be pseudo-patterned using proteins from another virus, provided that combining modified retroviral / lentiviral (e.g., SIV) RNA sequences and / or using codon-optimized gag-pol genes (e.g., from SIV) does not negatively affect the manufacturing titer of the vector (or even increase the vector titer) and / or affect the expression of the transfected gene (or even increase the expression of the transfected gene). Non-limiting examples of other proteins that can be used to pseudo-pattern the retroviral / lentiviral (e.g., SIV) vectors of the present invention include G-glycoprotein (G-VSV) from vesicular stomatitis virus and spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or modified forms thereof; proteins such as those described in UK Patent Applications Nos. 2,118,685.3 and 2,105,278.2, each of which is incorporated herein by reference in its entirety.

[0109] The retroviral / lentiviral (e.g., SIV) vector of the present invention further comprises Gag, Pol, and / or GagPol. Typically, Gag, Pol, and / or GagPol are derived from the desired retroviral / lentiviral (e.g., SIV) vector. As a non-limiting example, if the retroviral vector of the present invention is SIV, then Gag, Pol, and / or GagPol are typically derived from SIV.

[0110] Gag, Pol, and / or GagPol sequences can be codon-optimized. The inventors have previously demonstrated that the manufacturing titers of retroviral vectors containing codon-optimized Gag, Pol, and / or GagPol polymers from SIV are unexpectedly unaffected (see International Application PCT / GB2022 / 050524, which is incorporated herein by reference in its entirety). In fact, the inventors have previously demonstrated that the manufacturing titers of retroviral vectors containing codon-optimized Gag, Pol, and / or GagPol from SIV, pseudo-patterned with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxoviruses, may even be improved. This benefit of maintained / improved retroviral / lentiviral (e.g., SIV) vector yield can be combined with the benefits of this invention in providing maintained / enhanced transgenic expression and / or maintained / enhanced retroviral / lentiviral (e.g., SIV) RNA sequence integration to retroviral / lentiviral (e.g., SIV) vectors, while addressing potential safety risks of retroviral / lentiviral (e.g., SIV) vectors as described herein and improving the safety profile.

[0111] In the case of Gag, Pol, and / or GagPol, codon optimization is a technique for maximizing protein expression by improving the translation efficiency of the coding gene. Translation efficiency is improved through modification of the nucleic acid sequence. Codon optimization is routine in this technique, and designing codon-optimized forms of a given nucleic acid sequence is within the usual practice of those familiar with the technique. However, predicting the effects of codon optimization on other parameters is not straightforward. For example, as described herein, prior knowledge teaches that under normal manufacturing conditions (when the vector gene is plastid rather than the gag-pol gene is restrictive), codon optimization of the gag-pol gene typically reduces vector yield.

[0112] The retroviral / lentiviral (e.g., SIV) vector of the present invention may comprise a codon-optimized Gag protein, a codon-optimized Pol protein, a codon-optimized GagPol polymer, or a combination thereof. Therefore, the present invention provides a retroviral / lentiviral (e.g., SIV) vector comprising a codon-optimized Gag protein, wherein the codon-optimized Gag protein comprises or is composed 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 higher, and at most 100% sequence identity with SEQ ID NO: 9. Preferably, the present invention provides a retroviral vector comprising a codon-optimized Gag protein, wherein the codon-optimized Gag protein comprises or is composed of an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 9. This invention provides a retroviral vector comprising a codon-optimized Pol protein, wherein the codon-optimized Pol protein comprises or is composed 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 higher, and at most 100% sequence identity with SEQ ID NO: 10. Preferably, this invention provides a retroviral vector comprising a codon-optimized Pol protein, wherein the codon-optimized Pol protein comprises or is composed of an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 10.

[0113] GagPol is a polymeric protein that is processed to produce several smaller proteins within the viral particle. The degree of processing of GagPol or any constitutive protein within the retroviral / lentiviral (e.g., SIV) vector of the present invention, and therefore its presence and / or concentration, can vary over time.

[0114] Therefore, the retroviral / lentiviral (e.g., SIV) vector of the present invention may comprise one or more of the following proteins: p17, p27, p8, protease, p51, p15, and p31. One or more of these proteins may be combined with Gag, Pol, and / or GagPol. Preferably, the present invention provides a retroviral vector comprising the following proteins: p17, p27, p8, protease, p51, p15, and p31. Again, these proteins may be combined with Gag, Pol, and / or GagPol.

[0115] 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 higher, and at most 100%, sequence identity with 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 with SEQ ID NO: 2.

[0116] 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 higher, and at most 100% sequence identity with 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 with SEQ ID NO: 3.

[0117] 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 higher, and at most 100% sequence identity with 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 with SEQ ID NO: 4.

[0118] 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 higher, and at most 100% sequence identity with 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 with SEQ ID NO: 5.

[0119] 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 higher, and at most 100%, sequence identity with 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 with SEQ ID NO: 6.

[0120] 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 higher, and at most 100% sequence identity with 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 with SEQ ID NO: 7.

[0121] 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 higher, and at most 100%, sequence identity with 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 with SEQ ID NO: 8.

[0122] The retroviral / lentiviral (e.g., SIV) vector of the present invention may comprise: 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 with SEQ ID NO: 2 (as described above); 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 with SEQ ID NO: 3 (as described above); and 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 with SEQ ID NO: 2 ...). 4. An amino acid sequence having or consisting of at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity with SEQ ID NO: 5; a protease comprising or consisting of an amino acid sequence having or consisting of at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity with SEQ ID NO: 5; a p51 protein comprising or consisting of an amino acid sequence having or consisting of at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to 100% sequence identity with SEQ ID NO: 5; 6 having or consisting of an amino acid sequence that is at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or up to 100% sequence identical to SEQ ID NO: 7 (as described above); p15 protein containing or consisting of an amino acid sequence that is at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or up to 100% sequence identical to SEQ ID NO: 7 (as described above); and p31 protein containing or consisting of an amino acid sequence that is at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or up to 100% sequence identical to SEQ ID NO: 8 (as described above).

[0123] The retroviral / lentiviral (e.g., SIV) vector according to the present invention may be integrase-competent (IC). Alternatively, the retroviral / lentiviral (e.g., SIV) vector may be integrase-deficient (ID).

[0124] Retroviral / lentiviral (e.g., SIV) vectors, such as those of the present invention, can integrate into the genome of transduced cells and elicit persistent expression, making them suitable for transducing stem / precursor cells. In the lungs, several regenerative cell types have been identified as specific cell lineages responsible for maintaining the airways and alveoli. These cell types include basal cells and submucosal glandular cells in the upper airways, rod-shaped cells and neuroendocrine cells in the bronchiolar airways, bronchoalveolar stem cells in the terminal bronchioles, and type II lung cells in the alveoli. Therefore, and without being bound by theory, it is generally believed that these retroviral / lentiviral (e.g., SIV) vectors achieve long-term gene expression of the transfected gene by introducing the transfected gene into one or more long-lived airway epithelial cells or cell types, such as basal cells and submucosal glandular cells in the upper airway, rod cells and neuroendocrine cells in the bronchiolar airways, bronchoalveolar stem cells in the terminal bronchioles, and type II lung cells in the alveoli. As shown herein, the integration of the retroviral / lentiviral (e.g., SIV) vectors of the present invention, having modified retroviral / lentiviral (e.g., SIV) RNA sequences, into the target cell genome is unexpectedly without negative impact, and may even actually increase it.

[0125] Therefore, the retroviral / lentiviral (e.g., SIV) vectors of the present invention can transduce one or more cells or cell lines with regenerative potential in the lungs (including airways and respiratory tracts) to achieve long-term gene expression. For example, the retroviral / lentiviral (e.g., SIV) vectors can transduce basal cells, such as basal cells in the upper airway / respiratory tract. Basal cells play a central role in 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% (larger airways) to 6% (smaller airways).

[0126] The retroviral / lentiviral (e.g., SIV) vector of the present invention can be used for in vitro transduction of isolated and amplified stem / precursor cells, followed by administration to a patient. Preferably, the retroviral / lentiviral (e.g., SIV) vector of the present invention is used for in vivo transduction of lung (or airway / respiratory tract) cells.

[0127] The retroviral / lentiviral (e.g., SIV) vectors of the present invention exhibit significant resistance to shear forces, with only a slight reduction in transduction capacity when transported via clinically relevant delivery devices such as bronchoscopes, spray bottles, and nebulizers.

[0128] The retroviral / lentiviral (e.g., SIV) vectors of the present invention can achieve high transgenic gene expression levels, resulting in high expression levels (therapeutic doses) of therapeutic proteins. When administered to patients, the retroviral / lentiviral (e.g., SIV) vectors of the present invention typically provide high transgenic gene expression levels. The terms high expression and therapeutic expression are used interchangeably herein. Expression can be measured by any suitable method (qualitative or quantitative, preferably quantitative), and concentrations are given in any suitable unit of measurement, such as ng / ml or nM.

[0129] The expression of the transgenic gene of interest can be given relative to the expression of the corresponding endogenous (deficient) gene in the patient. Expression can be measured based on mRNA or protein expression. The expression of the transgenic gene of the present invention (such as the functional CFTR gene) can be quantified relative to endogenous genes, such as the endogenous (abnormal) CFTR gene, based on the mRNA copy per cell or any other suitable unit.

[0130] The expression levels of the transgenic genes and / or encoded therapeutic proteins of this invention can be measured as needed in lung tissue, epithelial liner fluid, and / or serum / plasma. Therefore, high and / or therapeutic expression levels can refer to concentrations in the lungs, epithelial liner fluid, and / or serum / plasma.

[0131] The retroviral / lentiviral (e.g., SIV) vectors of the present invention exhibit effective airway cell uptake, enhanced transgenic gene expression, and no loss of efficacy upon repeated administration. Therefore, the retroviral / lentiviral (e.g., SIV) vectors of the present invention can produce durable, reproducible, high-level expression in airway cells without inducing inappropriate immune responses.

[0132] The retroviral / lentiviral (e.g., SIV) vectors of the present invention achieve long-term transfected gene expression, resulting in long-term expression of therapeutic proteins. As described herein, the terms "long-term expression," "continuous expression," "persistent expression," and "lasting expression" are used interchangeably. According to the present invention, long-term expression means the expression of a therapeutic gene and / or protein, preferably at a therapeutic dose, lasting 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 longer. Preferably, long-term expression means 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 longer, more preferably at least 360 days, at least 450 days, at least 720 days, or longer. This long-term expression can be achieved through repeated doses or through a single dose.

[0133] Repeated doses may be administered twice daily, daily, twice weekly, weekly, monthly, every two months, every three months, every four months, every six months, annually, every two years, or for longer periods. Dosing may continue for the required duration, such as at least six months, at least one year, two years, three years, four years, five years, ten years, fifteen years, twenty years, or longer, until the patient's lifetime.

[0134] Preferably, the present invention relates to F / HN retroviral / lentiviral vectors containing promoters and transgenic genes, particularly SIV F / HN vectors.

[0135] Retroviral and lentiviral RNA sequences Each retroviral vector particle contains a retroviral RNA sequence. The retroviral RNA sequence contains an LTR component, the desired sequence incorporated into the particle, and a transgenic expression cartridge. As a non-limiting example, the retroviral RNA sequence may contain or consist of the following: a retroviral LTR component (typically R and U5 at the 5' end of the sequence (5' to 3' reads), and U3 and R at the 3' end of the sequence (5' to 3' reads)), the desired retroviral sequence incorporated into the retroviral particle, and a transgenic expression cartridge. The transgenic expression cartridge typically contains suitable enhancer / promoter components, transgenic cDNA, and post-transcriptional regulatory components. Preferably, the retroviral RNA sequence contains an SIV LTR component, the desired sequence incorporated into the particle, and a transgenic expression cartridge. As a non-limiting example, the SIVRNA sequence may include or consist of the following: SIV LTR components (R and U5 at the 5' end of the sequence (5' to 3' reads), and U3 and R at the 3' end of the sequence (5' to 3' reads)), the desired SIV sequence to be incorporated into the retroviral particle, and the transgenic expression cartridge.

[0136] The retroviral or lentiviral RNA sequences of the present invention are modified compared to unmodified retroviral or lentiviral RNA sequences derived therefrom. Modification of the retroviral or lentiviral RNA sequences compared to their derivatives provides advantageous properties. Non-limiting examples of such advantageous properties include maintained / enhanced transgenic gene expression compared to unmodified retroviral or lentiviral RNA sequences derived therefrom, maintained / enhanced integration of the retroviral / lentiviral (e.g., SIV) RNA sequence into the target / host cell genome, maintained / enhanced vector yield, and / or improved patient safety.

[0137] Compared to the derived retroviral or lentiviral RNA sequences, the modified retroviral or lentiviral RNA sequences of the present invention may feature codon substitutions and / or contain a reduced number of retroviral or lentiviral ORFs. For example, compared to the derived retroviral or lentiviral RNA sequences, the modified retroviral or lentiviral RNA sequences of the present invention may contain a reduced number of retroviral or lentiviral ORFs. Generally, compared to the derived retroviral or lentiviral RNA sequences, the modified retroviral or lentiviral RNA sequences of the present invention feature codon substitutions and contain a reduced number of retroviral or lentiviral ORFs.

[0138] 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. In the modified retroviral or lentiviral genome of the present invention, at least one, at least two, at least three, at least four, at least five, or more codons may be substituted. 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 of retroviral or lentiviral RNA sequences in this manner are known in this 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.

[0139] Introducing a stop codon causes premature termination of translation, resulting in an ORF of shorter length compared to the corresponding unmodified ORF without the stop sequence. Therefore, according to the present invention, retroviral or lentiviral RNA sequences are typically modified to introduce one or more stop codons, thereby reducing the length of one or more ORFs. For example, the length of one or more ORFs can be reduced by introducing UAG, UAA, or UGA codons into the retroviral RNA sequence (or TAG, TAA, or TGA codons into the original retroviral DNA sequence). As described herein, the stop codon can be removed by deleting or substituting nucleotides within the retroviral RNA sequence or the corresponding original retroviral DNA sequence, or by adding one or more (e.g., 1, 2, or 3) nucleotides to disrupt 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 the retroviral or lentiviral vector at the time of administration to a target. Therefore, the retroviral or lentiviral vectors of the present invention, containing modified retroviral or lentiviral RNA sequences, may have an improved safety profile compared to retroviral or lentiviral vectors containing unmodified retroviral or lentiviral RNA sequences derived from modified retroviral or lentiviral RNA sequences. As a non-limiting example, reducing the length of one or more retroviral or lentiviral ORFs reduces the risk of immune responses triggered by expression of longer polypeptides encoded by corresponding unmodified retroviral or lentiviral ORFs. Furthermore, as demonstrated herein, the length of one or more retroviral or lentiviral ORFs can be reduced without negatively impacting the expression of downstream transgenic genes, 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 the expression of downstream transgenic genes, retroviral or lentiviral vector integration, and / or retroviral or lentiviral vector yield.

[0140] As illustrated herein, such modifications may include or consist of the following: a modified retroviral or lentiviral RNA sequence is used to introduce a stop codon to reduce the length of one or more viral, particularly retroviral / lentiviral (e.g., SIV) ORFs in the 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 can be achieved by modifying the vector gene plasmid (i.e., pDNA1) used to generate the modified retroviral or lentiviral vector of the present invention, as described herein. Therefore, compared to the corresponding unmodified plasmid gene vector (i.e., pDNA1), the modified vector gene plasmid (i.e., pDNA1) may contain one or more ORFs of reduced length, particularly one or more retroviral / lentiviral (e.g., SIV) ORFs.

[0141] As a non-limiting example, the modified retroviral or lentiviral (e.g., SIV) RNA sequences of the present 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 stop codon typically reducing the length of the retroviral or lentiviral (e.g., SIV) ORF. Typically, the length of this one or more retroviral or lentiviral (e.g., SIV) ORF is reduced compared to the corresponding retroviral or lentiviral (e.g., SIV) ORF in an unmodified retroviral or lentiviral (e.g., SIV) RNA sequence from which the modified retroviral or lentiviral (e.g., SIV) RNA sequence is derived. Therefore, compared to the corresponding unmodified plastid genomic vector (i.e., pDNA1), the vector genomic plastid used to generate the modified retroviral or lentiviral (e.g., SIV) vector of the present invention may contain one or more ORFs of reduced length, particularly one or more retroviral / lentiviral (e.g., SIV) ORFs.

[0142] The 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 starting from the 5' of the transgenic gene and / or the transgenic gene promoter (also referred to as upstream). The length of one or more retroviral or lentiviral (e.g., SIV) ORFs starting from the 5' of the transgenic gene and / or the transgenic gene promoter may be reduced. As a non-limiting example, the length of at least one, at least two, at least three, at least four, at least five, or more retroviral or lentiviral (e.g., SIV) ORFs starting from the 5' of the transgenic gene and / or the transgenic gene activator may be reduced. Preferably, the length of one or two retroviral or lentiviral (e.g., SIV) ORFs at the 5' of the transgenic gene promoter may be reduced. 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. Therefore, compared to the corresponding unmodified plastid gene vector (i.e., pDNA1), the vector gene plastid used to generate the modified retroviral or lentiviral (e.g., SIV) vector of the present invention may contain one or more upstream ORFs of reduced length, especially one or more upstream retroviral / lentiviral (e.g., SIV) ORFs.

[0143] Introducing a stop codon can reduce the length of a polypeptide encoded by a retrovirus or lentivirus (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.

[0144] Alternatively, each introduced stop codon can reduce the length of one or more retroviral or lentiviral (e.g., SIV) ORFs encoding peptides of at least 10 amino acids, such as at least 50 amino acids, at least 100 amino acids, at least 200 amino acids, or longer, compared to the length of the unmodified ORF before the introduction of stop codons. For example, the introduction of stop codons can reduce the length of one or more retroviral or lentiviral (e.g., SIV) ORFs encoding peptides of at least 230 amino acids.

[0145] Therefore, as a non-limiting example, the introduction of a stop codon can reduce the length of a polypeptide encoded by a retroviral or lentiviral (e.g., SIV) ORF, wherein (i) the length of the polypeptide encoded by the (unmodified ORF) is at least 230 amino acids; and (ii) the length of the polypeptide encoded by the ORF is reduced by at least 40 amino acids or more.

[0146] Introducing individual stop codons can reduce the length of more than one ORF, especially one or more retroviral / lentiviral ORFs. In particular, introducing individual stop codons can reduce the length of two or three ORFs, especially two or three retroviral / ORFs, with a preferred reduction of two ORFs.

[0147] Other codon substitutions include the removal and / or replacement of one or more restriction enzyme sites. Such codon substitutions can be applied to generate the retroviral / lentiviral vectors of the present invention.

[0148] Preferred codon substitutions may comprise or consist of the following: a frameshift mutation and a stop codon substitution in the Env ORF of a retroviral / lentiviral RNA sequence. Such substitutions typically reduce the length of the Env ORF and prevent read-through from the Env ORF to the cPPT sequence. As illustrated, one such preferred codon substitution comprises 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 EnvORF from 235 amino acids to 192 amino acids, and also reduces the length of the polypeptide encoded by another retroviral / lentiviral ORF from 19 amino acids to 9 amino acids. The motif corresponding to residues 2354-2360 of SEQ ID NO: 1 is found at residues 1601-1607 of SEQ ID NO: 1.

[0149] Another preferred codon substitution, which could replace the preceding segment or be used otherwise, is the introduction of an Sbfl restriction site within the retroviral / lentiviral RNA sequence, which may optionally replace the EcoR1 restriction site. As illustrated, one such preferred codon substitution comprises 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: 1 is found at residues 985-993 of SEQ ID NO: 1.

[0150] Preferably, codon substitutions comprise or consist of the following combinations: (a) replacing a frameshift mutation and a stop codon in the Env ORF of a retroviral / lentiviral RNA sequence; and (b) introducing an Sbfl restriction site into the retroviral / lentiviral RNA sequence, which may optionally replace an EcoR1 restriction site. As illustrated, preferred codon substitutions comprise or consist of the following: (a) replacing a motif corresponding to residues 2347-2352 of SEQ ID NO: 25 with a motif corresponding to residues 2354-2360 of SEQ ID NO: 25; and (b) replacing a motif corresponding to residues 1734-1739 of SEQ ID NO: 25 with a motif corresponding to residues 1738-1746 of SEQ ID NO: 25.

[0151] Retroviral or lentiviral RNA sequences are typically modified to reduce the number of ORFs. For example, the number of ORFs can be reduced by removing the AUG codon from the retroviral RNA sequence (or the ATG codon from the original retroviral DNA sequence). As described herein, the start codon can be removed by deleting or substituting nucleotides within the start codon or by adding one or more (e.g., 1, 2, or 3) nucleotides to disrupt the start codon. Preferably, the retroviral or lentiviral RNA sequence is modified to reduce the number of retroviral or lentiviral ORFs. Removing one or more retroviral or lentiviral ORFs has the potential to improve the safety of the retroviral or lentiviral vector upon administration to a target. Therefore, the retroviral or lentiviral vector of the present invention, comprising a modified retroviral or lentiviral RNA sequence, may have an improved safety profile compared to a retroviral or lentiviral vector comprising an 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 immune responses triggered by the expression of those one or more retroviral or lentiviral ORFs. Furthermore, as demonstrated herein, removal of one or more retroviral or lentiviral ORFs can occur without negatively impacting the expression of downstream transgenic genes, the integration of retroviral or lentiviral vectors, and / or the yield of retroviral or lentiviral vectors. Removal of one or more retroviral or lentiviral ORFs can increase the expression of downstream transgenic genes, the integration of retroviral or lentiviral vectors, and / or the yield of retroviral or lentiviral vectors.

[0152] As illustrated herein, such modifications may include or consist of the following: modifying the retroviral or lentiviral RNA sequence to remove the virus, particularly retroviral / lentiviral (e.g., SIV) ORF, from 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 can be achieved by modifying the vector gene plasmid (i.e., pDNA1) used to generate the modified retroviral or lentiviral vector of the present invention, as described herein. Therefore, the modified vector gene plasmid (i.e., pDNA1) may contain a reduced number of viruses, particularly retroviral / lentiviral (e.g., SIV) ORF, compared to the corresponding unmodified plasmid gene vector (i.e., pDNA1). Thus, the modified retroviral or lentiviral vector of the present invention contains a reduced number of non-transgenic gene ORFs on its retroviral or lentiviral RNA sequence.

[0153] As a non-limiting example, the modified retroviral or lentiviral (e.g., SIV) RNA sequence of the present invention may be modified to remove at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more retroviral or lentiviral (e.g., SIV) ORFs, typically at least 6 or at least 7, preferably 6 or 7. Generally, 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. Therefore, the vector genome somatosome used to generate the modified retroviral or lentiviral (e.g., SIV) vector of the present invention may have a reduced number of retroviral or lentiviral (e.g., SIV) ORFs compared to the corresponding unmodified vector genome somatosome.

[0154] The retroviral or lentiviral (e.g., SIV) RNA sequence may be modified to reduce the number of retroviral or lentiviral (e.g., SIV) ORFs at the 5' (also referred to as upstream) of the transgene and / or transgene promoter. One or more retroviral or lentiviral (e.g., SIV) ORFs starting from the 5' of the transgene and / or transgene promoter may be removed. As a non-limiting example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more retroviral or lentiviral (e.g., SIV) ORFs starting from the 5' of the transgene and / or transgene activator may be removed, typically at least 6 or at least 7 retroviral or lentiviral (e.g., SIV) ORFs, preferably 6 or 7. Preferably, one or more retroviral or lentiviral (e.g., SIV) ORFs are removed from the 5' of the transgene promoter. Compared to an unmodified retroviral or lentiviral (e.g., SIV) RNA sequence from which a modified retroviral or lentiviral (e.g., SIV) RNA sequence is derived, the number of upstream ORFs can be reduced. Therefore, compared to the corresponding unmodified vector gene somatosome, the vector gene somatosome used to generate the modified retroviral or lentiviral (e.g., SIV) vector of the present invention can have a reduced number of upstream retroviral or lentiviral (e.g., SIV) ORFs.

[0155] Alternatively, according to the present invention, one or more retroviral or lentiviral (e.g., SIV) ORFs removed can each independently encode a polypeptide of length greater than or equal to 10 amino acids, greater than or equal to 20 amino acids, greater than or equal to 30 amino acids, greater than or equal to 40 amino acids, greater than or equal to 50 amino acids, greater than or equal to 60 amino acids, greater than or equal to 70 amino acids, greater than or equal to 80 amino acids, greater than or equal to 90 amino acids, greater than or equal to 100 amino acids, greater than or equal to 110 amino acids, greater than or equal to 120 amino acids, greater than or equal to 130 amino acids, greater than or equal to 140 amino acids, or greater than or equal to 150 amino acids. Typically, one or more retroviral or lentiviral (e.g., SIV) ORFs removed according to the present invention can each independently encode a polypeptide of length greater than or equal to 100 amino acids. Preferably, compared to an unmodified retroviral or lentiviral (e.g., SIV) RNA sequence from which a modified retroviral or lentiviral (e.g., SIV) RNA sequence is derived, at least one retroviral or lentiviral (e.g., SIV) ORF encoding a polypeptide of 100 or more amino acids in length can be removed from the modified retroviral or lentiviral (e.g., SIV) RNA sequence. Therefore, compared to an unmodified plastid genomic vector from which a modified retroviral RNA sequence is derived, the vector genomic plastid used to generate the modified retroviral or lentiviral (e.g., SIV) vector of the present invention can have one or more retroviral or lentiviral (e.g., SIV) ORFs encoding a polypeptide of 100 or more amino acids in length removed.

[0156] Therefore, compared to unmodified retroviral or lentiviral (e.g., SIV) RNA sequences that derive modified retroviral or lentiviral (e.g., SIV) RNA sequences, the retroviral or lentiviral (e.g., SIV) RNA sequences of the present invention may lack any ORF (other than the transgenic gene) encoding a polypeptide of 200 or more, 190 or more, 180 or more, 170 or more, or 160 or more amino acids in length. Therefore, compared to unmodified plastid genomic vectors that derive modified retroviral RNA sequences, the vector genomic plastids used to generate the modified retroviral or lentiviral (e.g., SIV) vectors of the present invention may lack any ORF (other than the transgenic gene) encoding a polypeptide of 200 or more amino acids in length as described above.

[0157] Compared to unmodified retroviral or lentiviral (e.g., SIV) RNA sequences that derive modified retroviral or lentiviral (e.g., SIV) RNA sequences, the retroviral or lentiviral (e.g., SIV) RNA sequences of the present invention may lack any ORF encoding a polypeptide of the following lengths within a portion of the Gag region: 180 amino acids or more, 100 amino acids or more, 90 amino acids or more, 80 amino acids or more, or 70 amino acids or more. Therefore, compared to unmodified plastid genomic vectors that derive modified retroviral RNA sequences, the vector genomic plastids used to generate the modified retroviral or lentiviral (e.g., SIV) vectors of the present invention may lack any ORF (except for transgenic genes) encoding a polypeptide of 180 amino acids or more in the portion of the Gag region described above.

[0158] Compared to unmodified retroviral or lentiviral (e.g., SIV) RNA sequences that derive modified retroviral or lentiviral (e.g., SIV) RNA sequences, the retroviral or lentiviral (e.g., SIV) RNA sequences of the present invention may lack any ORF encoding a polypeptide of 200 or more, 170 or more, or 160 or more amino acids in a portion of the RRE region. Therefore, compared to unmodified plastid genomic vectors that derive modified retroviral RNA sequences, the vector genomic plastids used to generate the modified retroviral or lentiviral (e.g., SIV) vectors of the present invention may lack any ORF (except for transgenic genes) encoding a polypeptide of 160 or more amino acids in a portion of the RRE region as described above.

[0159] Alternatively, one or more retroviral or lentiviral (e.g., SIV) ORFs to be removed may be contained (at least partially) within the RRE sequence. Preferably, one or more retroviral or lentiviral (e.g., SIV) ORFs are contained (at least partially) within a portion of the RRE sequence. Thus, compared to an unmodified retroviral or lentiviral (e.g., SIV) RNA sequence from which a modified retroviral or lentiviral (e.g., SIV) RNA sequence is derived, the retroviral or lentiviral (e.g., SIV) RNA sequence may be modified to reduce the number of ORFs contained (at least partially) within the portion of the RRE sequence. Thus, compared to an unmodified cytoplasmic vector from which a modified retroviral RNA sequence is derived, the vector cytoplasm 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) within the portion of the RRE sequence.

[0160] Alternatively, one or more retroviral or lentiviral (e.g., SIV) ORFs may be included (at least partially) in a portion of the Gag sequence. Therefore, compared to an unmodified retroviral or lentiviral (e.g., SIV) RNA sequence from which a modified retroviral or lentiviral (e.g., SIV) RNA sequence is derived, the retroviral or lentiviral (e.g., SIV) RNA sequence may be modified to reduce the number of ORFs included (at least partially) in a portion of the Gag sequence. Therefore, compared to an unmodified plasmosome vector from which a modified retroviral RNA sequence is derived, the vector plasmosome used to generate the modified retroviral or lentiviral (e.g., SIV) vector of the present invention may have a reduced number of ORFs included (at least partially) in a portion of the Gag sequence.

[0161] Unless explicitly stated otherwise, references herein to regions contained in retroviral / lentiviral (e.g., SIV) sequences, such as those contained in partial sequences, are considered valid. Gag Sequence or part RRE The reference to ORF in the sequence also applies, without reservation, to regions partially contained within the retroviral / lentiviral (e.g., SIV) sequence, such as those partially contained within... Gag Sequence or part RRE The ORF in the sequence. The ORF to be removed may extend across different regions of the retroviral / lentiviral (e.g., SIV) sequence and is therefore contained within two or more regions of the retroviral / lentiviral (e.g., SIV) sequence. For example, the ORF to be removed may extend across a portion Gag Sequence to Part RRE in sequence.

[0162] Generally, removing one or more retroviral or lentiviral (e.g., SIV) ORFs does not negatively affect the expression of downstream transgenic genes compared to unmodified retroviral or lentiviral (e.g., SIV) RNA sequences. Removing one or more retroviral or lentiviral (e.g., SIV) ORFs may increase the expression of downstream transgenic genes compared to unmodified retroviral or lentiviral (e.g., SIV) RNA sequences. Unmodified retroviral RNA sequences can be generated from the aforementioned unmodified plasmosome vectors.

[0163] Although modified retroviral RNA or lentiviral (e.g., SIV) sequences may not contain ORFs other than transgenic genes (especially retroviral or lentiviral (e.g., SIV) ORFs), this is not essential. Specifically, modified retroviral or lentiviral (e.g., SIV) RNA sequences may still contain ORFs other than transgenic genes (including retroviral or lentiviral (e.g., SIV) sequences, but may contain a reduced number of non-transgenic ORFs compared to unmodified retroviral or lentiviral (e.g., SIV) RNA sequences from which modified retroviral or lentiviral (e.g., SIV) RNA sequences are derived. Alternatively, the length of the remaining non-transgenic ORFs may be reduced compared to unmodified retroviral or lentiviral (e.g., SIV) RNA sequences from which modified retroviral or lentiviral (e.g., SIV) RNA sequences are derived. Therefore, the vector genomic plasmids used to generate the modified retroviral or lentiviral (e.g., SIV) vectors of the present invention may have a reduced number of non-transgenic ORFs compared to the derived unmodified plasmid genomic body (pDNA1). Alternatively, the length of the remaining non-transgenic ORF in the vector genome of the vector of the present invention can be reduced, compared with the unmodified retroviral or lentiviral (e.g., SIV) RNA sequence that derives the modified retroviral or lentiviral (e.g., SIV) RNA sequence.

[0164] Preferred modifications to reduce the number of ORFs, especially retroviral / lentiviral (e.g., SIV) ORFs, may include one or more of the following: (i) inserting nucleic acid (e.g., inserting U into the retroviral / lentiviral RNA sequence or inserting T into the corresponding proviral DNA sequence) to disrupt the start codon; (ii) replacing A in the retroviral / lentiviral RNA sequence with U (or replacing A in the corresponding proviral DNA sequence with T) to disrupt the start codon; and / or (iii) replacing U in the retroviral / lentiviral RNA sequence with A (or replacing T in the corresponding proviral DNA sequence with A) to disrupt the start codon.

[0165] As illustrated, preferred modifications to reduce the number of ORFs, particularly retroviral / lentiviral (e.g., SIV) ORFs, include: (i) introducing U into the retroviral / lentiviral RNA sequence or T into the corresponding proviral DNA sequence at 3' of residue 1183 immediately adjacent to SEQ ID NO: 25 (such insertion corresponds to residue 1184 of SEQ ID NO: 19 and residue 431 of SEQ ID NO: 1); (ii) introducing U into the retroviral / lentiviral RNA sequence or T into the corresponding proviral DNA sequence at 3' of residue 1287 immediately adjacent to SEQ ID NO: 25 (such insertion corresponds to residue 1289 of SEQ ID NO: 19 and residue 536 of SEQ ID NO: 1); (iii) introducing U into the retroviral / lentiviral RNA sequence or T into the corresponding proviral DNA sequence at 3' of residue 1303 immediately adjacent to SEQ ID NO: 25 (such insertion corresponds to residue 1306 of SEQ ID NO: 19 and residue 536 of SEQ ID NO: 19). (iv) Introducing U into the retroviral / lentiviral RNA sequence or T into the corresponding proviral DNA sequence at 3' of residue 1625 of SEQ ID NO: 25 (such insertion corresponds to residue 1629 of SEQ ID NO: 1 and residue 876 of SEQ ID NO: 1); (v) 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), replacing A in the retroviral / lentiviral RNA sequence with U or replacing A in the corresponding proviral DNA sequence with T; (vi) At residue 2064 of SEQ ID NO: 25 (corresponding to residue 2071 of SEQ ID NO: 19 and residue 1318 of SEQ ID NO: 1), replacing U in the retroviral / lentiviral RNA sequence with A or replacing T in the corresponding proviral DNA sequence with A; and / or (vii) At SEQ ID NO: At residue 2238 of 25 (corresponding to residue 2245 of SEQ ID NO: 19 and residue 1492 of SEQ ID NO: 1), U in the retroviral / lentiviral RNA sequence is replaced by A or T in the corresponding proviral DNA sequence is replaced by A.

[0166] Preferred modifications to reduce the number of ORFs, especially retroviral / lentiviral (e.g., SIV) ORFs, include combinations of the following or combinations thereof: (i) insertion of nucleic acids (e.g., insertion of U into the retroviral / lentiviral RNA sequence or insertion of T into 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 in the retroviral / lentiviral RNA sequence with U (or substitution of A in the corresponding proviral DNA sequence with T) to disrupt one or more start codons; and / or (iii) substitution of U in the retroviral / lentiviral RNA sequence with A (or substitution of T in the corresponding proviral DNA sequence with A) to disrupt one or more start codons (e.g., 2, 3, or 4, preferably 2 start codons).As illustrated, preferred modifications for removing one or more retroviral / lentiviral (e.g., SIV) ORFs include or consist of the following: (i) the introduction of U into the retroviral / lentiviral RNA sequence or T into the corresponding proviral DNA sequence at 3' of residue 1183 immediately adjacent to SEQ ID NO: 25 (such insertion corresponds to residue 1184 of SEQ ID NO: 19 and residue 431 of SEQ ID NO: 1); (ii) the introduction of U into the retroviral / lentiviral RNA sequence or T into the corresponding proviral DNA sequence at 3' of residue 1287 immediately adjacent to SEQ ID NO: 25 (such insertion corresponds to residue 1289 of SEQ ID NO: 19 and residue 536 of SEQ ID NO: 1); (iii) the introduction of U into the retroviral / lentiviral RNA sequence or T into the corresponding proviral DNA sequence at 3' of residue 1303 immediately adjacent to SEQ ID NO: 25 (such insertion corresponds to residue 1306 of SEQ ID NO: 19 and residue 536 of SEQ ID NO: 19). (iv) Introducing U into the retroviral / lentiviral RNA sequence or T into the corresponding proviral DNA sequence at 3' of residue 1625 of SEQ ID NO: 25 (such insertion corresponds to residue 1629 of SEQ ID NO: 1 and residue 876 of SEQ ID NO: 1); (v) 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), replacing A in the retroviral / lentiviral RNA sequence with U or replacing A in the corresponding proviral DNA sequence with T; (vi) At residue 2064 of SEQ ID NO: 25 (corresponding to residue 2071 of SEQ ID NO: 19 and residue 1318 of SEQ ID NO: 1), replacing U in the retroviral / lentiviral RNA sequence with A or replacing T in the corresponding proviral DNA sequence with A; and (vii) At SEQ ID NO: At residue 2238 of 25 (corresponding to residue 2245 of SEQ ID NO: 19 and residue 1492 of SEQ ID NO: 1), U in the retroviral / lentiviral RNA sequence is replaced by A or T in the corresponding proviral DNA sequence is replaced by A.

[0167] As a specific non-limiting example, modification of the modified retroviral or lentiviral (e.g., SIV) RNA sequence may remove (at least partially) the retroviral or lentiviral (e.g., SIV) ORF contained in a portion of the Gag region of the retroviral or lentiviral (e.g., SIV) RNA sequence, and / or may reduce the size of one or more retroviral or lentiviral (e.g., SIV) ORFs in that region. Preferably, the modified retroviral or lentiviral (e.g., SIV) RNA sequence of the present invention has been modified such that it does not contain any retroviral or lentiviral (e.g., SIV) ORF encoding a polypeptide of more than 100 amino acids, typically more than 70 amino acids, in a portion of the Gag region. Preferably, the modified retroviral or lentiviral (e.g., SIV) RNA sequence of the present invention has been modified such that it does not contain any retroviral or lentiviral (e.g., SIV) ORF encoding a polypeptide of more than 200 amino acids, typically more than 160 amino acids, in a portion of the RRE region. Preferably, the modified retroviral or lentiviral (e.g., SIV) RNA sequence of the present invention is modified such that it does not contain (i) any retroviral or lentiviral (e.g., SIV) ORF encoding a polypeptide of more than 100 amino acids, typically more than 70 amino acids, in a portion of the Gag region; and (ii) any retroviral or lentiviral (e.g., SIV) ORF encoding a polypeptide of more than 200 amino acids, typically more than 160 amino acids, in a portion of the RRE region. The present invention provides a retroviral or lentiviral (e.g., SIV) vector comprising the modified retroviral or lentiviral (e.g., SIV) RNA sequence.

[0168] Any modification or combination thereof used to reduce the number of ORFs within the retroviral or lentiviral (e.g., SIV) RNA sequence of the present invention, particularly the number of ORFs in retroviral or lentiviral (e.g., SIV) ORFs, may be used in combination with any codon substitution modification or combination thereof as described herein.

[0169] Therefore, the present invention provides a modified retroviral or lentiviral (e.g., SIV) RNA sequence comprising: (a) any retroviral or lentiviral (e.g., SIV) ORF that does not contain (i) a polypeptide encoding more than 100 amino acids, typically more than 70 amino acids, within a portion of the Gag region; (ii) any retroviral or lentiviral (e.g., SIV) ORF that encodes a polypeptide encoding more than 200 amino acids, typically more than 160 amino acids, within a portion of the RRE region; and (b) a codon substitution comprising or consisting of a combination of: (i) a frameshift mutation and a stop codon substitution in the EnvORF of the retroviral / lentiviral RNA sequence; and (ii) the introduction of an Sbfl restriction site in the retroviral / lentiviral RNA sequence, particularly in certain examples described herein, which may optionally substitute for the EcoR1 restriction site. The present invention provides a retroviral or lentiviral (e.g., SIV) vector comprising this modified retroviral or lentiviral (e.g., SIV) RNA sequence.

[0170] Any codon substitution or combination thereof may be used in conjunction with any modification or combination thereof that reduces the number of ORFs, especially retroviral / lentiviral (e.g., SIV) ORFs. The following retroviral / lentiviral (e.g., SIV) RNA sequences are preferred, wherein (a) the codon substitution comprises or consists of a combination of: (i) a frameshift mutation and a stop codon substitution in the Env of the retroviral / lentiviral RNA sequence. In the ORF; and (ii) introducing an Sbfl restriction site into the retroviral / lentiviral RNA sequence, which may optionally replace the EcoR1 restriction site; and (b) reducing the number of ORFs, especially retroviral / lentiviral (e.g., SIV) ORFs, comprising or consisting of combinations of the following: (i) inserting nucleic acids (e.g., inserting U into the retroviral / lentiviral RNA sequence or inserting T into the corresponding proviral DNA sequence) to disrupt one or more start codons (e.g., 2, 3 or 4, preferably 4 start codons); (ii) replacing A in the retroviral / lentiviral RNA sequence with U (or replacing A in the corresponding proviral DNA sequence with T) to disrupt one or more start codons; and (iii) replacing U in the retroviral / lentiviral RNA sequence with A (or replacing T in the corresponding proviral DNA sequence with A) to disrupt one or more start codons (e.g., 2, 3 or 4, preferably 2 start codons).

[0171] The following retroviral / lentiviral (e.g., SIV) RNA sequences are preferred, wherein (a) codon substitution comprises or consists of the following combinations: (i) 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: 25; and (ii) 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: 25; and (b) modifications that reduce the number of ORFs, particularly retroviral / lentiviral (e.g., SIV) ORFs, comprise or consist of the following combinations: (i) introducing U into the retroviral / lentiviral RNA sequence or introducing T into the corresponding proviral DNA sequence at 3' of residue 1183 immediately adjacent to SEQ ID NO: 25 (such insertion corresponds to residues 1184 of SEQ ID NO: 19 and SEQ ID NO: 19). (ii) The 3' of residue 1287 immediately adjacent to SEQ ID NO: 25 introduces U into the retroviral / lentiviral RNA sequence or T into the corresponding proviral DNA sequence (such insertion corresponds to residue 1289 of SEQ ID NO: 19 and residue 536 of SEQ ID NO: 1); (iii) The 3' of residue 1303 immediately adjacent to SEQ ID NO: 25 introduces U into the retroviral / lentiviral RNA sequence or T into the corresponding proviral DNA sequence (such insertion corresponds to residue 1306 of SEQ ID NO: 19 and residue 553 of SEQ ID NO: 1); (iv) The 3' of residue 1625 immediately adjacent to SEQ ID NO: 25 introduces U into the retroviral / lentiviral RNA sequence or T into the corresponding proviral DNA sequence (such insertion corresponds to residue 1629 of SEQ ID NO: 19 and residue 876 of SEQ ID NO: 1); (v) The 3' of residue 1787 of SEQ ID NO: 25 (corresponding to SEQ ID NO: 1) introduces U into the retroviral / lentiviral RNA sequence or T into the corresponding proviral DNA sequence (such insertion corresponds to residue 1629 of SEQ ID NO: 19 and residue 876 of SEQ ID NO: 1); (vi) At residue 1794 of SEQ ID NO: 19 and residue 1041 of SEQ ID NO: 1, replace A in the retroviral / lentiviral RNA sequence with U or replace A in the corresponding proviral DNA sequence with T; at residue 2064 of SEQ ID NO: 25 (corresponding to residue 2071 of SEQ ID NO: 19 and residue 1318 of SEQ ID NO: 1), replace U in the retroviral / lentiviral RNA sequence with A or replace T in the corresponding proviral DNA sequence with A;(vii) At residue 2238 of SEQ ID NO: 25 (corresponding to residue 2245 of SEQ ID NO: 19 and residue 1492 of SEQ ID NO: 1), the U in the retroviral / lentiviral RNA sequence is replaced by A, or the T in the corresponding proviral DNA sequence is replaced by A.

[0172] Preferably, the present invention provides an SIV vector pseudo-patterned with Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) protein, wherein: (a) the vector comprises a nucleic acid sequence containing SEQ ID NO: 1 or a modified retroviral RNA sequence composed of the nucleic acid sequence, preferably wherein the modified retroviral RNA sequence comprises the nucleic acid sequence of SEQ ID NO: 1; and (b) the F protein comprises a first unit containing an amino acid sequence containing SEQ ID NO: 14 or a second unit containing an amino acid sequence containing SEQ ID NO: 15 or a second unit composed of the amino acid sequence. The vector may further comprise one or more of the following: (a) p17 protein, comprising or consisting of the amino acid sequence of SEQ ID NO: 2; (b) p24 protein, comprising or consisting of the amino acid sequence of SEQ ID NO: 3; (c) p8 protein, comprising or consisting of the amino acid sequence of SEQ ID NO: 4; (d) protease, comprising or consisting of the amino acid sequence of SEQ ID NO: 5; (e) p51 protein, comprising or consisting of the amino acid sequence of SEQ ID NO: 6; (f) p15 protein, comprising or consisting of the amino acid sequence of SEQ ID NO: 7; (g) p31 protein, comprising or consisting of the amino acid sequence of SEQ ID NO: 8; (h) Gag protein, comprising or consisting of the amino acid sequence of SEQ ID NO: 9; and / or (i) Pol protein, comprising or consisting of the amino acid sequence of SEQ ID NO: 10. Optionally, the carrier comprises each of (a) to (g), and may further comprise one or both of (h) and (i).

[0173] The retroviral / lentiviral (e.g., SIV) RNA sequences of the present invention may contain one or more modifications other than codon substitutions and / or reductions in the retroviral / lentiviral (e.g., SIV) ORF as described herein. As a non-limiting example, the retroviral / lentiviral (e.g., SIV) RNA sequence may lack CpG (or contain no CpG) to promote gene expression. Standard techniques for modifying transgenic gene sequences in this manner are known in this art.

[0174] As illustrated herein, the retroviral / lentiviral (e.g., SIV) vectors of the present invention comprising modified retroviral / lentiviral (e.g., SIV) RNA sequences have at least maintained and potentially increased transgenic gene expression; and / or at least maintained and potentially increased integration of the retroviral / lentiviral (e.g., SIV) RNA sequences into target cells. Compared to unmodified retroviral / lentiviral (e.g., SIV) RNA sequences derived from modified retroviral / lentiviral (e.g., SIV) RNA sequences, the retroviral / lentiviral (e.g., SIV) vectors of the present invention comprising modified retroviral / lentiviral (e.g., SIV) RNA sequences generally also have at least maintained and potentially increased vector yield. This effect on vector yield can be further increased by using codon-optimized GagPol, as described herein.

[0175] Retroviral / lentiviral (e.g., SIV) vectors contain a promoter operatively linked to a transgenic gene, enabling expression of the transgenic gene. Typically, the promoter is a heterozygous human CMV enhancer / EF1a (hCEF) promoter. This hCEF promoter may lack introns corresponding to nucleotides 570-709 and exons corresponding to nucleotides 728-733 of the hCEF promoter. A preferred example of the hCEF promoter sequence of the present invention is provided by SEQ ID NO: 26. The promoter may be a CMV promoter. An example of a CMV promoter sequence is provided by SEQ ID NO: 27. The promoter may be a human elongation factor 1a (EF1a) promoter. An example of an EF1a promoter is provided by SEQ ID NO: 28. Other promoters for transgenic gene expression are known in the art, and their suitability for the retroviral / lentiviral (e.g., SIV) vectors of the present invention is determined using conventional techniques known in the art. Non-limiting examples of other promoters include UbC and UCOE. As described herein, the promoter can be modified to further regulate the expression of the transgenic gene of this invention.

[0176] The promoters included in the retroviral / lentiviral (e.g., SIV) vectors of the present invention can be specifically selected and / or modified to further optimize the regulation of the expression of therapeutic genes. Similarly, suitable promoters and standard techniques for their modification are known in this art. As a non-limiting example, various suitable (CpG-free) promoters applicable to the present invention are described in Pringle et al. (J. Mol. Med. Berl. 2012, 90(12):1487-96), which are incorporated herein by reference in their entirety. Preferably, the retroviral / lentiviral vectors of the present invention (especially SIV F / HN vectors) comprise an hCEF promoter having a low or no CpG dinucleotide content. The hCEF promoter may replace all CG dinucleotides with any of AG, TG, or GT. Thus, 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 generally further improves the efficacy of the retroviral / lentiviral (e.g., SIV) vectors of the present invention, especially when it is not necessary to induce an immune response against the expressed antigen or an inflammatory response against the delivered express construct. Elimination of CpG dinucleotides reduces the occurrence of influenza-like symptoms and inflammation, which may be caused by administration of the construct, especially when administered into the airway.

[0177] The retroviral / lentiviral (e.g., SIV) vectors of this invention can be modified to allow gene expression to be turned off. Standard techniques for modifying vectors in this manner are known in this art. As a non-limiting example, the Tet reactive promoter is widely used.

[0178] The retroviral / lentiviral (e.g., SIV) vector of the present invention may contain a transgenic gene encoding a polypeptide or protein that is therapeutic for treating such diseases, especially diseases or conditions of the airways, respiratory tract or lungs.

[0179] Therefore, the retroviral / lentiviral (e.g., SIV) vector of the present invention may comprise a transfection gene encoding a protein selected from: (i) a secreted therapeutic protein, optionally α-1 antitrypsin (A1AT), factor VIII, surfactant protein B (SFTPB), factor VII, factor IX, factor X, factor XI, Weinwidman's factor, granulocyte-macrophage community-stimulating factor (GM-CSF), and a monoclonal antibody against the vector; or (ii) CFTR, ABCA3, DNAH5, DNAH11, DNAI1 and DNAI2 Other examples of transgenic genes that may be included in the retroviral / lentiviral (e.g., SIV) vectors of the present invention include genes that are lacking or associated with other surfactants.

[0180] The transgenic gene included in the vector of the present invention can be modified to promote expression. For example, the transgenic gene sequence can be in a CpG-deficient (or CpG-free) form and / or further modified to promote gene expression. Standard techniques for modifying transgenic gene sequences in this manner are known in this art.

[0181] Preferably, the transgenic gene encodes CFTR. CFTR Examples of cDNA are provided by SEQ ID NO: 29. This also includes variants thereof (as described herein), particularly 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 higher, up to 100%, of identity with SEQ ID NO: 29. Preferably, the CTFR transgenic gene has at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 29.

[0182] The transgenic gene can encode A1AT. A1AT Examples of transgenic genes are provided by SEQ ID NO: 30 or by the complementary sequence of SEQ ID NO: 31. SEQ ID NO: 30 is a codon-optimized CpG-deficient A gene previously designed by the inventors to enhance translation in human cells. 1AT Transgenic genes. Such optimizations have been shown to enhance gene expression by up to 15-fold. This invention also covers unmodified (wild-type) genes. A1AT Variants of the same sequence (as defined herein) that have the same technical effect of enhancing translation compared to the gene sequence. A1AT The polypeptide encoded by the transgenic gene may be exemplified by the polypeptide of SEQ ID NO: 32. This also includes its variants (as described herein), particularly 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 higher, and at most 100%, of identity with SEQ ID NO: 30, 31, or 32. Preferably, the A1AT variant has at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 30, 31, or 32.

[0183] The transgenic gene can encode FVIII. FVIII Examples of transgenic genes are provided by the complementary sequences of SEQ ID NO: 33 and 34 or SEQ ID NO: 35 and 36. FVIIIThe polypeptide encoded by the transgenic gene may be exemplified by the polypeptide of SEQ ID NO: 37 or 38. Variants thereof (as described herein) are also included, particularly 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 higher, and at most 100%, of any of SEQ ID NO: 33 to 38. Preferably, the FVIII variant has at least 90%, at least 95%, or at least 99% identity with any of SEQ ID NO: 33 to 38.

[0184] The transgenic gene of this invention can be DNAH5 , DNAH11 , DNAI1 and DNAI2 Any one or more of them, or other known related genes.

[0185] When respiratory epithelium is targeted with retroviral / lentiviral (e.g., SIV) vectors, transgenic genes can encode A1AT, SFTPB, or GM-CSF. Transgenic genes can encode monoclonal antibodies (mAbs) against the vector. Transgenic genes can encode anti-TNF α. Transgenic genes can encode therapeutic proteins involved in inflammatory, immune, or metabolic conditions.

[0186] The retroviral / lentiviral (e.g., SIV) vectors of the present invention can be delivered to respiratory cells to allow the production of proteins to be secreted into the circulatory system. In such embodiments, the transgenic gene may encode factors VII, VIII, IX, X, XI, and / or Weinwidman's factor. Such vectors can be used to treat diseases, particularly cardiovascular diseases and hematologic disorders, preferably coagulation disorders such as hemophilia. Similarly, the transgenic gene may encode proteins involved in mAbs of the transgenic agent or in inflammatory, immune, or metabolic conditions such as cytotoxicity.

[0187] The retroviral / lentiviral (e.g., SIV) vectors of the present invention may not have introns located between the promoter and the transgenic gene. Similarly, in the vector gene body (pDNA1) plastid (e.g., pGM830 as described herein, having the sequence of SEQ ID NO: 20), there may be no introns between the promoter and the transgenic gene.

[0188] In some preferred embodiments, the retroviral / lentiviral (e.g., SIV) vector contains an hCEF promoter and a CFTR transfection gene, including those described herein. Optionally, the retroviral / lentiviral (e.g., SIV) vector may not have an intron located between the promoter and the transfection gene. Such retroviral / lentiviral (e.g., SIV) vectors can be used with the methods described herein, carrying... CFTR Genetic ploids of transgenic genes and promoters are produced.

[0189] In some preferred embodiments, the retroviral / lentiviral (e.g., SIV) vector contains an hCEF promoter and an A1AT transgenic gene, including those described herein. Optionally, the retroviral / lentiviral (e.g., SIV) vector may not have an intron located between the promoter and the transgenic gene. Such retroviral / lentiviral (e.g., SIV) vectors can be used with the methods described herein, carrying... A1AT Genetic ploids of transgenic genes and promoters are produced.

[0190] In some preferred embodiments, the retroviral / lentiviral (e.g., SIV) vector contains an hCEF or CMW promoter and an FVIII transgene, including those described herein. Optionally, the retroviral / lentiviral (e.g., SIV) vector may not have an intron located between the promoter and the transgene. Such retroviral / lentiviral (e.g., SIV) vectors can be used with the methods described herein, carrying... FVIII Genetic ploids of transgenic genes and promoters are produced.

[0191] Retroviral / lentiviral (e.g., SIV) vectors, as described herein, contain transgenic genes. These transgenic genes contain nucleic acid sequences encoding gene products (e.g., proteins, particularly therapeutic proteins).

[0192] For example, in one embodiment, encoding CFTR, A1AT or FVIII The nucleic acid sequence contains respectively with CFTR, A1AT or FVIII The nucleic acid sequence (examples of which are described herein) has 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 higher, and at most 100% sequence identity (or consists of such a nucleic acid sequence). In another embodiment, encoding CFTR , A1AT or FVIII The nucleic acid sequence contains respectively with CFTR, A1AT or FVIIII. A nucleic acid sequence (examples of which are described herein) having at least 95% (such as at least 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity (or consisting of such a nucleic acid sequence). In one embodiment, encoding CFTR The nucleic acid sequence 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 NO: 33 and 34 or by the complementary sequences of SEQ ID NO: 35 and 36 or their variants.

[0193] The amino acid sequence of the CFTR, A1AT, or FVIII transgenic gene may contain an amino acid sequence (or consist of) that is identical to the functional CFTR, A1AT, or FVIII polypeptide sequence by 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%.

[0194] The retroviral / lentiviral (e.g., SIV) vectors of the present invention may contain a central polypurine region (cPPT) and / or a marmot hepatitis virus posttranscriptional regulatory component (WPRE). An exemplary WPRE sequence is provided by SEQ ID NO: 39.

[0195] As described herein, retroviral / lentiviral (e.g., SIV) RNA sequences are derived from proviral DNA sequences. The proviral DNA sequence itself is provided during the manufacturing process via vector gene plasmid pDNA1. However, retroviral / lentiviral (e.g., SIV) RNA sequences are not identical to the proviral DNA sequence (and therefore not identical to vector gene plasmid pDNA1). Specifically, the length of retroviral / lentiviral (e.g., SIV) RNA sequences is shorter than the corresponding proviral DNA sequence, and the exact boundaries or limits of retroviral / lentiviral (e.g., SIV) RNA sequences are generally not easily determined. In other words, it is generally not possible to identify the exact retroviral / lentiviral (e.g., SIV) RNA sequence (specifically, the 5' and 3') solely from the proviral DNA sequence (and therefore the vector gene plasmid pDNA1 sequence).

[0196] Retroviral / lentiviral (e.g., SIV) vectors typically contain modified retroviral / lentiviral (e.g., SIV) RNA sequences of less than 10,000, 9,000, or 8,000 bases in length. Preferably, the retroviral / lentiviral (e.g., SIV) vector contains a modified retroviral / lentiviral (e.g., SIV) RNA sequence of less than 9,000 bases in length.

[0197] The retroviral / lentiviral (e.g., SIV) vector may contain or be composed of a modified retroviral / lentiviral (e.g., SIV) RNA 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 higher, and at most 100%, identical to SEQ ID NO: 1. The modified retroviral / lentiviral (e.g., SIV) RNA sequence may contain or be composed of a nucleic acid sequence having at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 1. The modified retroviral / lentiviral (e.g., SIV) RNA sequence may contain or be composed of a nucleic acid sequence having at least 99% identical to SEQ ID NO: 1. The modified retroviral sequence may contain or be composed of the nucleic acid sequence of SEQ ID NO: 1.

[0198] This invention provides a retroviral / lentiviral (e.g., SIV) vector comprising a retroviral / lentiviral (e.g., SIV) RNA 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 higher, and at most 100% identical to the nucleic acid sequence of SEQ ID NO: 1. Modified retroviral / lentiviral (e.g., SIV) RNA sequences may consist of nucleic acid sequences having at least 90%, at least 95%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 1. Modified retroviral / lentiviral (e.g., SIV) RNA sequences may consist of nucleic acid sequences having at least 99% identical to the nucleic acid sequence of SEQ ID NO: 1. This invention provides a retroviral / lentiviral (e.g., SIV) vector comprising a retroviral / lentiviral (e.g., SIV) RNA sequence composed of the nucleic acid sequence of SEQ ID NO: 1.

[0199] The retroviral / lentiviral (e.g., SIV) vector may contain the following modified retroviral / lentiviral (e.g., SIV) RNA sequence, which (a) is less than 10,000 bases, less than 9,000 bases, or less than 8,000 bases in length; and (b) contains or consists of a nucleic acid sequence that is 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 higher, and at most 100% identical to SEQ ID NO: 1.

[0200] The retroviral / lentiviral (e.g., SIV) vector may contain the following modified retroviral / lentiviral (e.g., SIV) RNA sequence, which (a) is less than 10,000 bases, less than 9,000 bases, or less than 8,000 bases in length; and (b) contains 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 higher, and at most 100% identical to SEQ ID NO: 1.

[0201] The retroviral / lentiviral (e.g., SIV) vector may contain the following modified retroviral / lentiviral (e.g., SIV) RNA sequence, which (a) is less than 10,000 bases, less than 9,000 bases, or less than 8,000 bases in length; and (b) contains or consists of a nucleic acid sequence having at least 99%, at least 99.5%, at least 99.9%, or higher, and at most 100% identity with SEQ ID NO: 1.

[0202] The retroviral / lentiviral (e.g., SIV) vector may contain the following modified retroviral / lentiviral (e.g., SIV) RNA sequence, which (a) is less than 10,000 bases, less than 9,000 bases, 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 higher, and at most 100% identical to SEQ ID NO: 1.

[0203] The retroviral / lentiviral (e.g., SIV) vector may contain the following modified retroviral / lentiviral (e.g., SIV) RNA sequence, which (a) is less than 10,000 bases, less than 9,000 bases, 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 higher, and at most 100%, identical to SEQ ID NO: 1.

[0204] The retroviral / lentiviral (e.g., SIV) vector may contain the following modified retroviral / lentiviral (e.g., SIV) RNA sequence, which is (a) less than 10,000 bases, less than 9,000 bases, 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 higher, and at most 100% identical to SEQ ID NO: 1.

[0205] The retroviral / lentiviral (e.g., SIV) vector may contain the following modified retroviral / lentiviral (e.g., SIV) RNA sequence, which (a) is less than 9,000 bases or less than 8,000 bases in length; and (b) contains 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 higher, and at most 100% identical to, SEQ ID NO: 1.

[0206] The retroviral / lentiviral (e.g., SIV) vector may contain the following modified retroviral / lentiviral (e.g., SIV) RNA sequence, which (a) is less than 9,000 bases or less than 8,000 bases in length; and (b) contains 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 higher, and at most 100% identical to, SEQ ID NO: 1.

[0207] The retroviral / lentiviral (e.g., SIV) vector may contain the following modified retroviral / lentiviral (e.g., SIV) RNA sequence, which (a) is less than 9,000 bases or less than 8,000 bases in length; and (b) contains or consists of a nucleic acid sequence having at least 99%, at least 99.5%, at least 99.9% or higher, and at most 100% identical to SEQ ID NO: 1.

[0208] The retroviral / lentiviral (e.g., SIV) vector may contain the following modified retroviral / lentiviral (e.g., SIV) RNA sequence, which (a) is less than 9,000 bases 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 higher, and at most 100% identical to SEQ ID NO:1.

[0209] The retroviral / lentiviral (e.g., SIV) vector may contain the following modified retroviral / lentiviral (e.g., SIV) RNA sequence, which (a) is less than 9,000 bases 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 higher, and at most 100% identical to SEQ ID NO:1.

[0210] A retroviral / lentiviral (e.g., SIV) vector may contain a modified retroviral / lentiviral (e.g., SIV) RNA sequence that is (a) less than 9,000 bases 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 higher, and at most 100% identical to SEQ ID NO:1.

[0211] Preferably, the retroviral / lentiviral (e.g., SIV) vector comprises the following modified retroviral / lentiviral (e.g., SIV) RNA sequence, wherein (a) it is less than 9,000 bases in length; and (b) it comprises or is composed 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 with SEQ ID NO: 1. More preferably, the retroviral / lentiviral (e.g., SIV) vector comprises the following modified retroviral / lentiviral (e.g., SIV) RNA sequence, wherein (a) it is less than 9,000 bases in length; and (b) it comprises or is composed of a nucleic acid sequence having at least 99% identity with SEQ ID NO: 1. More preferably, the retroviral / lentiviral (e.g., SIV) vector comprises the following modified retroviral / lentiviral (e.g., SIV) RNA sequence, which (a) is less than 9,000 bases in length; and (b) consists of a nucleic acid sequence having at least 99% identity with SEQ ID NO: 1. More preferably, the retroviral / lentiviral (e.g., SIV) vector comprises the following modified retroviral / lentiviral (e.g., SIV) RNA sequence, which (a) is less than 9,000 bases in length; and (b) comprises or consists of the nucleic acid sequence of SEQ ID NO: 1. More preferably, the retroviral / lentiviral (e.g., SIV) vector comprises the following modified retroviral / lentiviral (e.g., SIV) RNA sequence, which (a) is less than 9,000 bases in length; and (b) consists of the nucleic acid sequence of SEQ ID NO: 1.

[0212] The 5' and / or 3' boundaries of the modified retroviral / lentiviral (e.g., SIV) RNA sequence can each independently allow some 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.

[0213] Therefore, for example, compared to SEQ ID NO: 1, the modified retroviral / lentiviral (e.g., SIV) RNA sequence may contain up to 200 additional nucleotides, up to 150 additional nucleotides, up to 100 additional nucleotides, up to 75 additional nucleotides, up to 50 additional nucleotides, up to 25 additional nucleotides, up to 10 additional nucleotides, or up to 5 additional 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 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 additional nucleotide at the 5' and / or 3' end. The presence and number of extra nucleotides at the 5' end of the modified retroviral / lentiviral (e.g., SIV) RNA sequence are independent of the presence and number of extra nucleotides at the 3' end. As a non-limiting example, for instance, compared to SEQ ID NO: 1, the modified retroviral / lentiviral (e.g., SIV) RNA sequence may contain up to 3 extra nucleotides at the 5' end and up to 200 extra nucleotides at the 3' end. As another non-limiting example, for instance, compared to SEQ ID NO: 1, the modified retroviral / lentiviral (e.g., SIV) RNA sequence may not contain any extra nucleotides at the 5' end but may contain up to 42 extra nucleotides at the 3' end. Preferably, for instance, compared to SEQ ID NO: 1, the modified retroviral / lentiviral (e.g., SIV) RNA sequence does not contain any extra nucleotides at the 5' end but may contain up to 200 extra nucleotides at the 3' end (as described above).

[0214] Modified retroviral / lentiviral (e.g., SIV) RNA sequences may contain, for example, up to 200 nucleotides, up to 150 nucleotides, up to 100 nucleotides, up to 75 nucleotides, up to 50 nucleotides, up to 25 nucleotides, up to 10 nucleotides, or up to 5 nucleotides fewer than those in SEQ ID NO: 1 at the 5' and / or 3' ends. Modified retroviral / lentiviral (e.g., SIV) RNA sequences may contain, for example, 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 fewer than those in SEQ ID NO: 1 at the 5' and / or 3' ends. The number of deletions 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. As a non-limiting example, a modified retroviral / lentiviral (e.g., SIV) RNA sequence may contain, for example, up to 3 fewer nucleotides at the 5' end compared to SEQ ID NO: 1 and up to 200 fewer nucleotides at the 3' end compared to SEQ ID NO: 1. As another non-limiting example, a modified retroviral / lentiviral (e.g., SIV) RNA sequence may contain, for example, the same number of nucleotides at the 5' end compared to SEQ ID NO: 1 and up to 42 fewer nucleotides at the 3' end compared to SEQ ID NO: 1. Preferably, for example, compared to SEQ ID NO: 1, the modified retroviral / lentiviral (e.g., SIV) RNA sequence contains the same number of nucleotides at the 5' end, but may contain up to 200 fewer nucleotides at the 3' end (as described above).

[0215] The modified retroviral / lentiviral (e.g., SIV) RNA sequence may have additional nucleotides at one end, for example, compared to SEQ ID NO: 1, and fewer nucleotides at the other end, for example, compared to SEQ ID NO: 1. Therefore, the 5' end may have additional nucleotides, for example, compared to SEQ ID NO: 1, and the 3' end may have fewer nucleotides, for example, compared to SEQ ID NO: 1. The disclosure herein regarding the number of additional and / or missing nucleotides also applies, without reservation, to modified retroviral / lentiviral (e.g., SIV) RNA sequences having additional nucleotides at one end, for example, compared to SEQ ID NO: 1, and fewer nucleotides at the other end, for example, compared to SEQ ID NO: 1. Preferably, for example, compared to SEQ ID NO: 1, the modified retroviral / lentiviral (e.g., SIV) RNA sequence does not contain any extra / deleted nucleotides at the 5' end, but may contain extra or fewer nucleotides at the 3' end (as described above).

[0216] 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 transgenic gene expression; (ii) maintaining or even increasing the integration of retroviral / lentiviral (e.g., SIV) RNA sequences into the host cell genome; and / or (iii) maintaining or even increasing retroviral / lentiviral (e.g., SIV) vector yield.

[0217] Therefore, retroviral / lentiviral (e.g., SIV) vectors containing the modified retroviral / lentiviral (e.g., SIV) RNA sequences of the present invention typically exhibit high transgenic gene expression levels. Generally, 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 transgenic gene expression to retroviral / lentiviral (e.g., SIV) vectors containing unmodified retroviral / lentiviral (e.g., SIV) RNA sequences derived from the modified retroviral / lentiviral (e.g., SIV) RNA sequences (i.e., corresponding to unmodified retroviral / lentiviral (e.g., SIV) RNA sequences).

[0218] As used herein, the term "equivalent transgenic expression" can be defined such that a modified retroviral / lentiviral (e.g., SIV) RNA sequence does not significantly reduce transgenic expression of a retroviral / lentiviral (e.g., SIV) vector compared to its corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence. As a non-limiting example, transgenic expression in the host / target cell genome of a retroviral / lentiviral (e.g., SIV) vector containing a modified retroviral / lentiviral (e.g., SIV) RNA sequence may be no more than 2-fold, no more than 1.5-fold, no more than 1.0-fold, no more than 0.5-fold, no more than 0.25-fold, or lower than the transgenic expression of a retroviral / lentiviral (e.g., SIV) vector containing its corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence. The term “equivalent transgenic expression” can be defined such that the transgenic expression of a retroviral / lentiviral (e.g., SIV) vector containing a modified retroviral / lentiviral (e.g., SIV) RNA sequence in the host / target cell genome is statistically invariant (e.g., p < 0.05, p < 0.01) compared to the transgenic expression of a retroviral / lentiviral (e.g., SIV) vector containing the corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence.

[0219] Preferably, the transgenic expression of a retroviral / lentiviral (e.g., SIV) vector containing a modified retroviral / lentiviral (e.g., SIV) RNA sequence is increased in the host / target cell genome compared to the transgenic expression of a vector containing a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence. The transgenic expression of a retroviral / lentiviral (e.g., SIV) vector containing a modified retroviral / lentiviral (e.g., SIV) RNA sequence in the host / target cell genome may be at least 1.5 times, at least 2 times, or at least 2.5 times greater than the transgenic expression of a vector containing a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence.

[0220] Alternatively, retroviral / lentiviral (e.g., SIV) vectors containing the modified retroviral / lentiviral (e.g., SIV) RNA sequences of the present invention exhibit high vector integration rates into the host / target cell genome. Generally, 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 integration into the host / target cell genome to retroviral / lentiviral (e.g., SIV) vectors containing the corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequences.

[0221] As used herein, the term "equivalent integration" can be defined such that a modified retroviral / lentiviral (e.g., SIV) RNA sequence does not significantly reduce the integration of a 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, the integration of a retroviral / lentiviral (e.g., SIV) vector comprising the modified retroviral / lentiviral (e.g., SIV) RNA sequence of the present invention into the host / target cell genome may be no more than 2-fold, no more than 1.5-fold, no more than 1.0-fold, no more than 0.5-fold, no more than 0.25-fold, or lower 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” can be defined such that the integration of a retrovirus / lentivirus (e.g., SIV) vector containing the modified retrovirus / lentivirus (e.g., SIV) RNA sequence of the present invention into the host / target cell genome is statistically invariant (e.g., p<0.05, p<0.01) compared to the integration of a retrovirus / lentivirus (e.g., SIV) vector containing the corresponding unmodified retrovirus / lentivirus (e.g., SIV) RNA sequence.

[0222] Preferably, the integration of retroviral / lentiviral (e.g., SIV) vectors containing the modified retroviral / lentiviral (e.g., SIV) RNA sequence vectors of the present invention into the host / target cell genome is increased compared to the integration of retroviral / lentiviral (e.g., SIV) vectors containing the corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence vectors of the present invention into the host / target cell genome may be at least 1.5 times, at least 2 times, or at least 2.5 times higher than the integration of retroviral / lentiviral (e.g., SIV) vectors containing the corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence vectors.

[0223] Alternatively, the present invention provides highly potent purified retroviral / lentiviral (e.g., SIV) vectors comprising modified retroviral / lentiviral (e.g., SIV) RNA sequences. Typically, the potency of a retroviral / lentiviral (e.g., SIV) vector having the modified retroviral / lentiviral (e.g., SIV) RNA sequences of the present invention is at least equivalent to the potency of a retroviral / lentiviral (e.g., SIV) vector comprising the corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequences.

[0224] As used herein, the term "equivalent potency" can be defined such that a modified retroviral / lentiviral (e.g., SIV) RNA sequence does not significantly reduce the potency of a retroviral / lentiviral (e.g., SIV) vector compared to its corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence. As a non-limiting example, the potency of a retroviral / lentiviral (e.g., SIV) vector incorporating the modified retroviral / lentiviral (e.g., SIV) RNA sequence of the present invention may be no more than 2-fold, no more than 1.5-fold, no more than 1.0-fold, no more than 0.5-fold, no more than 0.25-fold, or lower than the potency of a retroviral / lentiviral (e.g., SIV) vector incorporating its corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence. The term "equivalent potency" can be defined such that the potency of a retrovirus / lentivirus (e.g., SIV) vector containing the modified retrovirus / lentivirus (e.g., SIV) RNA sequence of the present invention is statistically invariant (e.g., p<0.05, p<0.01) compared to the potency of a retrovirus / lentivirus (e.g., SIV) vector containing the corresponding unmodified retrovirus / lentivirus (e.g., SIV) RNA sequence.

[0225] Preferably, the potency of a retrovirus / lentivirus (e.g., SIV) vector containing the modified retrovirus / lentivirus (e.g., SIV) RNA sequence vector of the present invention is higher than that of a retrovirus / lentivirus (e.g., SIV) vector containing the corresponding unmodified retrovirus / lentivirus (e.g., SIV) RNA sequence. The potency of the retrovirus / lentivirus (e.g., SIV) vector containing the modified retrovirus / lentivirus (e.g., SIV) RNA sequence of the present invention may be at least 1.5 times, at least 2 times, or at least 2.5 times higher than that of a retrovirus / lentivirus (e.g., SIV) vector containing the corresponding unmodified retrovirus / lentivirus (e.g., SIV) RNA sequence.

[0226] The production of high-titer retroviral / lentiviral (e.g., SIV) vectors can impart additional desired properties to the resulting vector products. For example, without being bound by theory, it is believed that high-titer production, without the need for vigorous concentration via methods such as TFF, results in higher quality vector products compared to corresponding retroviral / lentiviral (e.g., SIV) vectors with unmodified retroviral / lentiviral (e.g., SIV) RNA sequences, because these vectors are exposed to less shearing forces that could damage viral particles and their RNA carriers.

[0227] Preferably, compared to the titer of a retroviral / lentiviral (e.g., SIV) vector containing a modified retroviral / lentiviral (e.g., SIV) RNA sequence vector of the present invention, the retroviral / lentiviral (e.g., SIV) vector exhibits maintained / enhanced transgenic gene expression. Compared to the titer of a retroviral / lentiviral (e.g., SIV) vector containing a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence vector, the retroviral / lentiviral (e.g., SIV) vector containing a modified retroviral / lentiviral (e.g., SIV) RNA sequence vector of the present invention exhibits maintained / enhanced transgenic gene expression and maintained / enhanced vector integration. Compared to the potency of retroviral / lentiviral (e.g., SIV) vectors containing corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequences, retroviral / lentiviral (e.g., SIV) vectors containing the modified retroviral / lentiviral (e.g., SIV) RNA sequence vectors of the present invention exhibit maintained / enhanced transgenic gene expression and maintained / enhanced vector yield / potency, respectively. More preferably, compared to the potency of retroviral / lentiviral (e.g., SIV) vectors containing corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequences, retroviral / lentiviral (e.g., SIV) vectors containing the modified retroviral / lentiviral (e.g., SIV) RNA sequence vectors of the present invention exhibit maintained / enhanced transgenic gene expression, maintained / enhanced vector integration, and maintained / enhanced vector yield / potency, respectively.

[0228] This invention also provides host cells comprising the retroviral / lentiviral (e.g., SIV) vector of this invention. Typically, 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 generating viruses are also readily available (as described herein).

[0229] Generation method The method for generating the retroviral / lentiviral (e.g., SIV) vectors of the present invention is also described herein.

[0230] The inventors have previously demonstrated that using the codon-optimized gal-pol gene from SIV does not negatively affect the manufacturing titer of SIV vectors pseudo-patterned from hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxoviruses, and may even improve the vector titer. This description is in PCT / GB2022 / 050524, which is incorporated herein by reference in its entirety.

[0231] The inventors hereby demonstrate that retroviral / lentiviral (e.g., SIV) vectors having modified retroviral / lentiviral (e.g., SIV) RNA sequences can be generated, avoiding the potential safety risks described herein, while: (i) maintaining or even increasing transgenic gene expression; (ii) maintaining or even increasing the 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. Furthermore, the vector genomic plasmids used to manufacture the retroviral / lentiviral (e.g., SIV) vectors of the present invention can be combined with the use of a codon-optimized gag-pol gene as described herein, while simultaneously maintaining or even increasing vector titer.

[0232] Therefore, the present invention provides a method for generating a retroviral / lentiviral (e.g., SIV) vector comprising a modified retroviral / lentiviral (e.g., SIV) RNA sequence as described herein, wherein the retroviral / lentiviral (e.g., SIV) is pseudo-patterned with hemagglutinin-neuraminidase (HN) and fusion (F) protein from respiratory paramyxovirus, and the sequence comprises a promoter and a transfection gene. Preferably, the retroviral / lentiviral (e.g., SIV) vector is a lentiviral vector, particularly a simian immunodeficiency virus (SIV) vector.

[0233] The method of the present invention can be a scalable GMP-compatible method.

[0234] The methods of this invention generally allow for the generation of retroviral / lentiviral (e.g., SIV) vectors containing modified retroviral / lentiviral (e.g., SIV) RNA sequences with high transgenic gene expression levels. Typically, the methods of this invention produce retroviral / lentiviral (e.g., SIV) vectors having modified retroviral / lentiviral (e.g., SIV) RNA sequences as described herein, which are at least equivalent in terms of transgenic gene expression to retroviral / lentiviral (e.g., SIV) vectors generated by the same method containing unmodified retroviral / lentiviral (e.g., SIV) RNA sequences derived from the modified retroviral / lentiviral (e.g., SIV) RNA sequences (i.e., corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequences).

[0235] As used herein, the term "equivalent transgenic expression" can be defined such that a modified retroviral / lentiviral (e.g., SIV) RNA sequence does not significantly reduce transgenic expression of a retroviral / lentiviral (e.g., SIV) vector compared to its corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence. As a non-limiting example, transgenic expression in the host / target cell genome of a retroviral / lentiviral (e.g., SIV) vector containing a modified retroviral / lentiviral (e.g., SIV) RNA sequence is not more than 2-fold, not more than 1.5-fold, not more than 1.0-fold, not more than 0.5-fold, not more than 0.25-fold, or lower than that of a retroviral / lentiviral (e.g., SIV) vector containing its corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence. The term “equivalent transgenic expression” can be defined such that the transgenic expression of a retroviral / lentiviral (e.g., SIV) vector containing a modified retroviral / lentiviral (e.g., SIV) RNA sequence in the host / target cell genome is statistically invariant (e.g., p < 0.05, p < 0.01) compared to the transgenic expression of a retroviral / lentiviral (e.g., SIV) vector containing the corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence produced by the same method.

[0236] Preferably, the transgenic expression of a retroviral / lentiviral (e.g., SIV) vector containing a modified retroviral / lentiviral (e.g., SIV) RNA sequence vector, generated by the same method, is increased in the host / target cell genome compared to the transgenic expression of a vector containing a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence vector. The transgenic expression of a transgenic gene containing a modified retroviral / lentiviral (e.g., SIV) RNA sequence vector in the host / target cell genome may be at least 1.5 times, at least 2 times, or at least 2.5 times greater than the transgenic expression of a vector containing a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence vector generated by the same method.

[0237] The methods of the present invention generally allow for the production of retroviral / lentiviral (e.g., SIV) vectors containing modified retroviral / lentiviral (e.g., SIV) RNA sequences with high integration levels into the host / target cell genome. Typically, the methods of the present invention produce retroviral / lentiviral (e.g., SIV) vectors having modified retroviral / lentiviral (e.g., SIV) RNA sequences as described herein, which are at least equivalent in integration into the host / target cell genome to retroviral / lentiviral (e.g., SIV) vectors produced by the same method containing corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequences.

[0238] As used herein, the term "equivalent integration" can be defined such that a modified retroviral / lentiviral (e.g., SIV) RNA sequence does not significantly reduce the integration of a 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, the integration of a retroviral / lentiviral (e.g., SIV) vector containing a modified retroviral / lentiviral (e.g., SIV) RNA sequence into the host / target cell genome may be no more than 2-fold, no more than 1.5-fold, no more than 1.0-fold, no more than 0.5-fold, no more than 0.25-fold, or less than the integration of a retroviral / lentiviral (e.g., SIV) vector containing the corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence into the host / target cell genome. The term “equivalent integration” can be defined such that the integration of a retrovirus / lentivirus (e.g., SIV) vector containing a modified retrovirus / lentivirus (e.g., SIV) RNA sequence into the host / target cell genome is statistically invariant (e.g., p < 0.05, p < 0.01) compared to the integration of a retrovirus / lentivirus (e.g., SIV) vector containing the corresponding unmodified retrovirus / lentivirus (e.g., SIV) RNA sequence produced by the same method.

[0239] Preferably, the integration of retroviral / lentiviral (e.g., SIV) vectors containing modified retroviral / lentiviral (e.g., SIV) RNA sequences into the host / target cell genome is increased compared to the integration of retroviral / lentiviral (e.g., SIV) vectors containing corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequences produced by the same method. The integration of retroviral / lentiviral (e.g., SIV) vectors containing modified retroviral / lentiviral (e.g., SIV) RNA sequences 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 the integration of retroviral / lentiviral (e.g., SIV) vectors containing corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequences produced by the same method.

[0240] The methods of this invention generally allow for the production of highly potent purified retroviral / lentiviral (e.g., SIV) vectors containing modified retroviral / lentiviral (e.g., SIV) RNA sequences. Typically, the methods of this invention produce retroviral / lentiviral (e.g., SIV) vectors having the modified retroviral / lentiviral (e.g., SIV) RNA sequences as described herein, with a potency at least equivalent to that of retroviral / lentiviral (e.g., SIV) vectors containing corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequences produced by corresponding methods.

[0241] As used herein, the term "equivalent potency" can be defined such that a modified retroviral / lentiviral (e.g., SIV) RNA sequence does not significantly reduce the potency of a retroviral / lentiviral (e.g., SIV) vector compared to its corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence. As a non-limiting example, the potency of a retroviral / lentiviral (e.g., SIV) vector containing a modified retroviral / lentiviral (e.g., SIV) RNA sequence may be no more than 2-fold, 1.5-fold, 1.0-fold, 0.5-fold, 0.25-fold, or lower than that of a retroviral / lentiviral (e.g., SIV) vector containing its corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence. The term “equivalent potency” can be defined such that the potency of a retrovirus / lentivirus (e.g., SIV) vector containing a modified retrovirus / lentivirus (e.g., SIV) RNA sequence is statistically invariant (e.g., p < 0.05, p < 0.01) compared to the potency of a retrovirus / lentivirus (e.g., SIV) vector containing a corresponding unmodified retrovirus / lentivirus (e.g., SIV) RNA sequence produced by the same method.

[0242] Preferably, the titer of a retroviral / lentiviral (e.g., SIV) vector containing a modified retroviral / lentiviral (e.g., SIV) RNA sequence is higher than that of a retroviral / lentiviral (e.g., SIV) vector containing a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence produced by the same method. The titer of the retroviral / lentiviral (e.g., SIV) vector containing the modified retroviral / lentiviral (e.g., SIV) RNA sequence may be at least 1.5 times, at least 2 times, or at least 2.5 times higher than that of a retroviral / lentiviral (e.g., SIV) vector containing a corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequence produced by the same method.

[0243] The generation of retroviral / lentiviral (e.g., SIV) vectors typically employs one or more plastids that provide the components required for vector generation: the retroviral / lentiviral vector genome, Gag-Pol, Rev, F, and HN. Multiple components can be provided on a single plastid. Preferably, each component is provided on a separate plastid, resulting in five plastids, one for each of the vector genome, Gag-Pol, Rev, F, and HN.

[0244] Alternatively, a single plastid can provide the Gag-Pol and Rev components and may be referred to as the packaging plastid (pDNA2). The remaining components (genome, F, and HN) can be provided by separate plastids (pDNA1, pDNA3a, and pDNA3b, respectively), such that four plastids are used to generate the retroviral / lentiviral (e.g., SIV) vector according to the invention. In the four-plastid approach, pDNA1, pDNA3a, and pDNA3b may be as described herein in the context of the five-plastid approach.

[0245] In the preferred pDNA1 method of this invention, the vector gene plastomer encodes all genetic material, including the transgenic gene, packaged in the final retroviral / lentiviral vector. The vector gene plastomer may be referred to herein as “pDNA1” and typically contains the transgenic gene and its promoter. As described herein, only a portion of the genetic material found in the vector gene plastomer ultimately enters the virus, and the exact boundaries and limits of this portion cannot be easily deduced from the first-order sequence of pDNA1. This invention elucidates for the first time a modified RNA sequence of a SIV vector that addresses many potential safety risks while maintaining or even increasing (i) transgenic gene expression, (ii) SIV RNA sequence integration, and / or (iii) vector yield.

[0246] The other four plasmids are manufacturing plasmids encoding the Gag-Pol, Rev, F, and HN proteins. These plasmids can be named "pDNA2a", "pDNA2b", "pDNA3a", and "pDNA3b", respectively.

[0247] Typically, lentiviruses are SIVs, such as SIV1, with SIV-AGM being the preferred choice. The F and HN proteins are derived from respiratory paramyxoviruses, with Sendai virus being the preferred choice.

[0248] In CFTR In one specific embodiment, the five plasmids are composed of Figures 1A to 1F Characterization, therefore pDNA1 is Figure 1A pGM830 plastids, pDNA2a is Figure 1B pGM691 plastid or Figure 1C pGM297 plastid, pDNA2b is Figure 1D pGM299 plastid, pDNA3a is Figure 1E pGM301 plastid and pDNA3b is Figure 1F The pGM303 plastid, or any variant of such plastids (as described herein). pGM326 (as... Figure 1G (As shown in the image) is an unmodified vector gene somatoform derived from pGM830.

[0249] When the method of this invention is used to produce A1AT, the five plastids can be obtained from... Figure 2 (Therefore, plasmid pDNA1 can be pGM407) and Figure 1B or Figure 1C and Figures 1D to 1F Characterized by all of them (as described above for a specific CFTR embodiment) or variants of any of these plastids (as described herein).

[0250] When the method of this invention is used to produce FVIII, the five plastids can be obtained from... Figures 3A to 3D One of them (therefore, plasmid pDNA1 can be pGM411, pGM412, pGM413, or pGM414) and Figure 1B or Figure 1C and Figures 1D to 1F Characterized by all of them, or by variations of any of these plastids (as described herein).

[0251] As in Figure 1A The plasmid defined in the document is represented by SEQ ID NO: 19; as in Figure 1B The plasmid defined in the document is represented by SEQ ID NO: 20; as in Figure 1C The plasmid defined in the document is represented by SEQ ID NO: 21; as in Figure 1D The plasmid defined in the document is represented by SEQ ID NO: 22; as in Figure 1E The plasmid defined in the document is represented by SEQ ID NO: 23; as in Figure 1F The plasmid defined in the document is represented by SEQ ID NO: 24; as in Figure 1G The plasmid defined in the document is represented by SEQ ID NO: 25; as in Figure 2 The plasmid defined in the document is represented by SEQ ID NO: 40; and as in Figures 3A to 3D The plastids 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 as defined herein are represented by SEQ ID NO: 41 to 44, respectively. This invention also covers variants of these plastids (as defined herein). In particular, it covers variants having at least 90% (such as at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) sequence identity with any of SEQ ID NO: 19 to 25 and 40 to 44.

[0252] In the five-plastosome method of this invention, all five plastosomes contribute to the formation of the final retroviral / lentiviral (e.g., SIV) vector, but only the vector gene plastosome provides the nucleic acid sequence contained in the retroviral / lentiviral (e.g., SIV) RNA sequence. During the fabrication of the retroviral / lentiviral (e.g., SIV) vector, the vector gene plastosome (pDNA1) provides the enhancer / promoter, Psi, RRE, cPPT, mWPRE, SIN LTR, SV40 polyadenylate (see [link to original text]). Figure 1A This is crucial for virus manufacturing. Using pGM830 as a non-limiting example of pDNA1, the CMV enhancer / promoter, SV40 polyadenylate, colE1 Ori, and KanR are involved in the manufacturing of retroviral / lentiviral (e.g., SIV) vectors (e.g., vGM195 or vGM244) of the present invention, but were not found in the final retroviral / lentiviral (e.g., SIV) vector. RREs, central polypurine region (cPPT), hCEF, soCFTR2 (transgenic gene), and mWPRE from pGM326 or pGM830 are found in the final retroviral / lentiviral (e.g., SIV) vector. Long terminal repeats (SIN / IN) self-inactivating (SINLTR) and packaging signals (Psi) are found in the final retroviral / lentiviral (e.g., SIV) vector.

[0253] For other retroviral / lentiviral (e.g., SIV) vectors of the present invention, the corresponding component from the gene somatoform (pDNA1) of other vectors is required for manufacturing (but not found in the final vector) or is present in the final retroviral / lentiviral (e.g., SIV) vector.

[0254] The F and HN proteins from pDNA3a and pDNA3b (preferably Sendai virus F and HN proteins) are crucial for infecting target cells with the final retroviral / lentiviral (e.g., SIV) vector, specifically for entry into patient epithelial cells (typically lung or nasal cells as described herein). The products of pDNA2a and pDNA2b plastids are essential for viral transduction, i.e., for inserting retroviral / lentiviral (e.g., SIV) DNA into the host genome. Promoters, regulatory components (such as WPRE), and transfected genes are crucial for transfected gene expression within target cells.

[0255] The method of the present invention may include or consist of the following steps: (a) cell suspension growth; (b) cell transfection with one or more plastids; (c) addition of nuclease; (d) collection of lentivirus (e.g., SIV); (e) addition of trypsin; and (f) purification of lentivirus (e.g., SIV).

[0256] This method can use the tetraplast or pentaplast system described herein. Therefore, for the preferred pentaplast method, one or more plastids may comprise or consist of the following: vector gene plastid pDNA1; co-galpol plastid (e.g., codon-optimized gagpol plastid) pDNA2a; Rev plastid pDNA2b; fusion (F) protein plastid pDNA3a; and hemagglutinin-neuraminidase (HN) plastid 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.

[0257] Any suitable ratio of vector gene plasmid:gagpol plasmid:Rev plasmid:F plasmid:HN plasmid can be used to further optimize (improve) the resulting retroviral / lentiviral (e.g., SIV) titer. As a non-limiting example, the ratio of vector gene plasmid:gagpol plasmid:Rev plasmid:F plasmid:HN plasmid can be in the range of 10-40:-4-20:3-12:3-12:3-12, typically 15-20:7-11:4-8:4-8:4-8, such as approximately 18-22:7-11:4-8:4-8:4-8, or 19-21:8-10:5-7:5-7:5-7. Preferably, the ratio of vector gene plasmid:gagpol plasmid:Rev plasmid:F plasmid:HN plasmid is approximately 20:9:6:6:6.

[0258] Steps (a)-(f) of the method are typically performed sequentially, beginning at step (a) and continuing up to step (f). The method may include one or more additional steps, such as additional purification steps, buffer exchange, post-purification concentration of the retroviral / lentiviral (e.g., SIV) vector, and / or post-purification (or concentration) formulation of the retroviral / lentiviral (e.g., SIV) vector. Each of these steps may contain one or more sub-steps. For example, collection may involve one or more steps or sub-steps, and / or purification may involve one or more steps or sub-steps.

[0259] Any suitable cell type can be transfected with one or more plastids (e.g., the five plastids described herein) to produce the retroviral / lentiviral (e.g., SIV) vector of the present invention. Mammalian cells, particularly human cell lines, are typically used. Non-limiting examples of cells suitable for 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 virus production cells – catalog number A35347, from ThermoFisher Scientific).

[0260] Cells can be grown in animal-free culture media, including serum-free media. Cells can be grown in culture media containing human components. Cells can be grown in defined culture media containing synthetically produced components or composed of them.

[0261] Any suitable transfection method can be used according to the present invention. The selection of a suitable transfection method is within the scope of conventional practice for those generally skilled in the art. As a non-limiting example, transfection can be performed using PEIPro. TM Lipofectamine 2000 TM Or Lipofectamine 3000 TM conduct.

[0262] Any suitable nuclease can be used according to the present invention. The selection of a suitable nuclease is within the scope of conventional practice for those generally 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 performed in the pre-collection stage, the post-collection stage, or between collection steps.

[0263] The gag-pol gene used to generate the retroviral / lentiviral (e.g., SIV) vector of the present invention can be codon-optimized. Therefore, the gag-pol gene within the pDNA2a plastid can 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 higher sequence identity with SEQ ID NO: 17, preferably having at least 95% identity with SEQ ID NO: 17. The codon-optimized gag-pol gene may consist of the nucleic acid sequence of SEQ ID NO: 17. Preferably, pDNA2a pGM691 comprises the codon-optimized gag-pol gene of SEQ ID NO: 17.

[0264] Gag-pol genes, including codon-optimized gag-pol genes (e.g., SIV gag-pol genes), are typically operatively linked to a promoter to facilitate the expression of the gag-pol protein. Any suitable promoter can be used, including those described herein in the context of promoters for transgenic genes. Preferably, the promoter is a CAG promoter, as used on the exemplary pGM691 plasmid. An exemplary CAG promoter is listed in SEQ ID NO: 45. The codon-optimized gag-pol gene of SEQ ID NO: 17 contains translational sliding and therefore does not form a single, conventional open reading frame.

[0265] Codon-optimized gag-pol genes (or nucleic acids containing or composed of them) and plasmids containing these genes or nucleic acids are advantageous in generating retroviral / lentiviral (e.g., SIV) vectors using the methods of the present invention because they allow for the generation of high-titer F / HN retroviral / lentiviral (e.g., SIV) vectors. Typically, these codon-optimized gag-pol genes (or nucleic acids containing or composed of them) and plasmids containing these genes or nucleic acids can be used to generate retroviral / lentiviral (e.g., SIV) vectors with titers at least equivalent to those generated by methods not using the codon-optimized gag-pol genes as described herein. Therefore, the use of codon-optimized gag-pol genes can be combined with modified retroviral / lentiviral (e.g., SIV) RNA sequences to further maintain / enhance vector titers.

[0266] The codon-optimized gag-pol gene was further revealed in PCT / GB2022 / 050524, which is incorporated herein by reference in its entirety.

[0267] The present invention also provides a retroviral / lentiviral (e.g., SIV) vector that can be obtained by the method of the present invention.

[0268] Typically, retroviral / lentiviral (e.g., SIV) vectors obtained by the methods of this invention are produced with high titers as described herein. Titer can be measured according to transduction units as defined herein. As described herein, the methods of this invention generally produce retroviral / lentiviral (e.g., SIV) vectors containing modified retroviral / lentiviral (e.g., SIV) RNA sequences, with titers equivalent to or higher than those containing corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequences and / or methods that do not use codon-optimized gag-pol genes.

[0269] Therefore, the retroviral / lentiviral (e.g., SIV) vectors of the present invention, including vectors obtainable by the methods of the present invention, may optionally have a density of at least about 2.5 × 10⁻⁶. 6 TU / mL, at least about 3.0 × 10 6 TU / mL, at least approximately 3.1 × 10⁻⁶ 6 TU / mL, at least about 3.2 × 10 6 TU / mL, at least about 3.3 × 10 6 TU / mL, at least approximately 3.4 × 10⁻⁶ 6 TU / mL, at least approximately 3.5 × 10 6 TU / mL, at least about 3.6 × 10 6TU / mL, at least approximately 3.7 × 10⁻⁶ 6 TU / mL, at least approximately 3.8 × 10⁻⁶ 6 TU / mL, at least approximately 3.9 × 10 6 TU / mL, at least about 4.0 × 10 6 A titer of TU / mL or higher. Preferably, the retroviral / lentiviral (e.g., SIV) vector has a titer of at least about 3.0 × 10⁻⁶. 6 TU / mL or at least about 3.5 × 10⁻⁶ 6 The potency of TU / mL is generated.

[0270] The production of high-titer retroviral / lentiviral (e.g., SIV) vectors can impart additional desired properties to the resulting vector products. For example, without being bound by theory, it is believed that high-titer production of retroviral / lentiviral (e.g., SIV) vectors, without the need for drastic concentration using methods such as TFF, results in higher quality vector products because these vectors are exposed to less shearing forces that could damage viral particles and their RNA carriers.

[0271] Typically, the gag-pol gene used (e.g., a codon-optimized gag-pol gene) is matched to the resulting retroviral / lentiviral vector. As a non-limiting example, when 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, when the lentiviral vector is an SIV vector, the codon-optimized gag-pol gene used is the SIV gag-pol gene.

[0272] Preferably, the codon-optimized gag-pol gene used is the SIV gag-pol gene.

[0273] As described herein, the retroviral / lentiviral (e.g., SIV) vectors of the present invention comprise modified retroviral / lentiviral (e.g., SIV) RNA sequences, which are typically modified to reduce the number of retroviral / lentiviral (e.g., SIV) ORFs. Therefore, the vector gene plasmids used to generate the retroviral / lentiviral (e.g., SIV) vectors of the present invention can be modified to reduce the number of retroviral / lentiviral (e.g., SIV) ORFs. All disclosures herein regarding modifications to the retroviral / lentiviral (e.g., SIV) RNA sequences, including modifications to reduce the number of retroviral / lentiviral (e.g., SIV) ORFs within the retroviral / lentiviral (e.g., SIV) RNA sequences, apply equally and without reservation to the vector gene plasmids (pDNA1) described herein, which can be used to generate the retroviral / lentiviral (e.g., SIV) vectors of the present invention.

[0274] As used herein, the term "trypsin" refers to trypsin and its equivalents. An equivalent enzyme is an enzyme that has the same or substantially the same cleavage specificity as trypsin. Trypsin cleavage activity can be defined as the cleavage of the C-terminus of an arginine or lysine residue, typically only at the C-terminus. Trypsin activity can preferably be provided by recombinant enzymes without animal origin, such as TrypLESelect™. Trypsin can be added pre-collection, post-collection, or between collection steps.

[0275] Any suitable purification method can be used to purify retroviral / lentiviral (e.g., SIV) vectors. Non-limiting examples of suitable purification steps include deep / end filtration, tangential flow filtration (TFF), and chromatography. Purification steps typically include at least one chromatographic step. Non-limiting examples of chromatographic steps that can be used according to the invention include mixed-mode size exclusion chromatography (SEC) and / or anion exchange chromatography. Dissociation can be performed with or without a salt gradient, preferably without a salt gradient.

[0276] This method can be used to generate the retroviral / lentiviral (e.g., SIV) vectors of the present invention, such as those comprising as described herein. CFTR , A1AT and / or FVIII A gene vector. Alternatively, the retroviral / lentiviral (e.g., SIV) vector of the present invention contains any of the above-mentioned genes or genes encoding the above-mentioned proteins.

[0277] This method can be used Figures 1A to 1F , Figure 2 and / or Figures 3A to 3D Any combination of one or more of the specific plasmid constructs provided is used to provide the retroviral / lentiviral (e.g., SIV) vector of the present invention. In particular, it is preferred to use... Figure 1A, Figure 2 or Figures 3A to 3D The use of plasmid combination Figure 1B and Figures 1D to 1F The material structure, in which Figure 1A Its quality is particularly good.

[0278] This invention also provides a method for improving the titer of retroviral / lentiviral (e.g., SIV) vectors, comprising using a modified retroviral / lentiviral (e.g., SIV) RNA sequence as described herein, or a vector gene plastid derived from such a modified retroviral / lentiviral (e.g., SIV) RNA sequence. This method can be combined with plastids containing a codon-optimized gag-pol gene (or nucleic acid containing or composed of it), as described herein, to further improve the titer of retroviral / lentiviral (e.g., SIV) vectors. Compared to methods that use corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequences or vector gene plastids derived from corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequences, the method of increasing the titer of retroviral / lentiviral (e.g., SIV) vectors according to the present invention can increase the titer by at least 1.5 times, at least 2 times, or at least 2.5 times or more, and optionally also uses an uncodon-optimized form of the gag-pol gene (or nucleic acid containing or composed of it), or plastids or host cells containing these uncodon-optimized gag-pol genes or nucleic acids. Alternatively, compared to methods using corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequences or vector gene plasmids derived from corresponding unmodified retroviral / lentiviral (e.g., SIV) RNA sequences, the method according to the invention for increasing retroviral / lentiviral (e.g., SIV) titers can increase titers by at least about 25%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, or more, and optionally also uses an uncodon-optimized form of the gag-pol gene (or nucleic acid containing or composed of it), or plasmids containing such uncodon-optimized genes or nucleic acids. Preferably, the method according to the invention for increasing retroviral / lentiviral (e.g., SIV) vector titers can increase titers 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%. Generally, the corresponding method is consistent with the method of the present invention, except that it uses a vector gene plasmid corresponding to an unmodified retroviral / lentiviral (e.g., SIV) RNA sequence or a vector gene plasmid derived from an unmodified retroviral / lentiviral (e.g., SIV) RNA sequence, and optionally a codon-optimized gag-pol gene (or nucleic acid containing or composed of it), and plasmids containing these genes or nucleic acids. All disclosures herein regarding methods for generating retroviral / lentiviral (e.g., SIV) vectors are equally and unreservedly applicable to the method of the present invention for improving the titer of retroviral / lentiviral (e.g., SIV).

[0279] This invention also provides the use of the modified retroviral / lentiviral (e.g., SIV) RNA sequence of the present invention (or vector gene plasmids derived from the modified retroviral / lentiviral (e.g., SIV) RNA sequence) for improving the titer of retroviral / lentiviral (e.g., SIV) vectors. This use can be combined with the use of codon-optimized gag-pol genes (or nucleic acids containing or composed of them), and plasmids containing these genes or nucleic acids as described herein, to further improve the titer of retroviral / lentiviral (e.g., SIV) vectors. Compared to the use of the modified retroviral / lentiviral (e.g., SIV) RNA sequence (or a vector gene plastid derived from the modified retroviral / lentiviral (e.g., SIV) RNA sequence) and optionally the corresponding uncodon-optimized form of the gag-pol gene (or nucleic acid containing or composed of it), or plastids containing these uncodon-optimized genes or nucleic acids, this use can increase the titer of retroviral / lentiviral (e.g., SIV) vectors by at least 1.5 times, at least 2 times, or at least 2.5 times or more. Alternatively, compared to the use of the modified retroviral / lentiviral (e.g., SIV) RNA sequence of the present invention (or a vector gene plastid derived from the modified retroviral / lentiviral (e.g., SIV) RNA sequence) and optionally the corresponding uncodon-optimized form of the gag-pol gene (or nucleic acid containing or composed of it), or plastids containing these uncodon-optimized genes or nucleic acids, this use can increase the retroviral / lentiviral (e.g., SIV) vector titer by at least about 25%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, or more. Preferably, this 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%. Generally, the corresponding applications are consistent with the methods of the present invention, except that the modified retroviral / lentiviral (e.g., SIV) RNA sequence of the present invention (or a vector gene plastid from which the unmodified retroviral / lentiviral (e.g., SIV) RNA sequence is derived) and optionally, a codon-optimized gag-pol gene (or nucleic acid containing or composed of it) and plastids containing the nucleic acids of these genes are also present.All disclosures herein regarding methods for generating retroviral / lentiviral (e.g., SIV) vectors are equally and without reservation applicable to the use of modified retroviral / lentiviral (e.g., SIV) RNA sequences (or vector gene plastids from which the modified retroviral / lentiviral (e.g., SIV) RNA sequences) and optionally codon-optimized gag-pol genes (or nucleic acids containing or composed of such genes), and plastids containing such genes or nucleic acids, to improve the potency of retroviral / lentiviral (e.g., SIV) vectors according to the invention.

[0280] The use of a combination of the codon-optimized gag-pol gene with the modified retroviral / lentiviral (e.g., SIV) RNA sequence of the present invention, or a vector gene plasmid derived from the modified retroviral / lentiviral (e.g., SIV) RNA sequence, can provide another advantage in terms of safety and / or vector titer. Therefore, the increased vector yield described herein can be achieved using a combination of the modified retroviral / lentiviral (e.g., SIV) RNA sequence of the present invention (or a vector gene plasmid derived from the modified retroviral / lentiviral (e.g., SIV) RNA sequence) with the codon-optimized gag-pol gene. This document relates to any and all disclosures regarding the increase in vector titer in the case of using the modified retroviral / lentiviral (e.g., SIV) RNA sequence of the present invention (or vector gene plasmids from which the modified retroviral / lentiviral (e.g., SIV) RNA sequence is derived), which are equally and without reservation applicable to the method of combining the modified retroviral / lentiviral (e.g., SIV) RNA sequence of the present invention (or vector gene plasmids from which the modified retroviral / lentiviral (e.g., SIV) RNA sequence is derived) with the codon-optimized gag-pol gene, and the vectors produced by such methods.

[0281] Treatment indications The retroviral / lentiviral (e.g., SIV) vectors of the present invention achieve high and durable gene expression through efficient gene transfer, while also reducing the risk of side effects attributable to retroviral ORFs, such as upstream ORFs. The F / HN pseudomodeling retroviral / lentiviral (e.g., SIV) vectors of the present invention are capable of: (i) airway transduction without disrupting epithelial integrity; (ii) persistent gene expression; (iii) no chronic toxicity; and (iv) efficient repeat dosing. Long-term / persistent stable gene expression, preferably at a therapeutically effective dose, can be achieved using repeated doses of the vectors of the present invention. Alternatively, a single dose can be used to achieve the desired long-term expression.

[0282] Therefore, advantageously, the retroviral / lentiviral (e.g., SIV) vectors of the present invention can be used in gene therapy. For example, the efficient airway cell absorption characteristics of the retroviral / lentiviral (e.g., SIV) vectors of the present invention make them highly suitable for treating respiratory 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 another example, the present invention provides the secretion of therapeutic proteins into the respiratory lumen or circulatory system. Therefore, the administration of the retroviral / lentiviral (e.g., SIV) vectors of the present invention and their absorption by airway cells enable the use of the lungs (or nose or airways) as a "factory" to produce therapeutic proteins, which are then secreted and enter the systemic circulation in therapeutic amounts, traveling in the circulation to the cells / tissues of interest to induce a therapeutic effect. Unlike intracellular or membrane proteins, the production of such secreted proteins is independent of transduced disease-specific target cells, which is a significant advantage and achieves high protein expression levels. Therefore, other diseases besides respiratory diseases, such as cardiovascular diseases and blood disorders, especially coagulation disorders, can also be treated with the retroviral / lentiviral (e.g., SIV) vector of the present invention.

[0283] The retroviral / lentiviral (e.g., SIV) vectors of the present invention can effectively treat diseases by providing transgenic genes for disease correction. For example, inserting... CFTR The functional replication of genes to improve or prevent lung disease in CF patients is independent of underlying mutations. Therefore, the retroviral / lentiviral (e.g., SIV) vectors of the present invention can be used to treat cystic fibrosis (CF), typically through gene therapy via CFTR transgenic genes as described herein.

[0284] As another example, the retroviral / lentiviral (e.g., SIV) vector of the present invention can be used to treat α-1 antitrypsin (A1AT) deficiency, typically via gene therapy using A1AT transgenic genes as described herein. A1AT is a secreted antiprotease, primarily produced in the liver and subsequently 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 to patients with A1AT deficiency and other lung diseases such as CF or chronic obstructive pulmonary disease (COPD) and offers the opportunity to overcome some of the problems encountered with known enzyme replacement therapies (where A1AT is isolated from human blood and administered intravenously weekly), thereby providing stable, durable performance in the target tissue (lung / nasal epithelium), ease of administration, and unlimited availability.

[0285] Transduction using the retroviral / lentiviral (e.g., SIV) vector of the present invention can induce the secretion of recombinant proteins into the lung lumen and circulation. One benefit of this is the therapeutic protein reaching the interstitium. Therefore, A1AT gene therapy may also be beneficial for other disease indications, 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 disease (GvH), multiple sclerosis, liver disease, cirrhosis, vasculitis, and infections such as bacterial and / or viral infections.

[0286] A1AT has many other anti-inflammatory and tissue-protective effects, such as in preclinical models of diabetes, graft-versus-host disease, and inflammatory bowel disease. Therefore, the transduction of A1AT in the lungs and / or nose according to the invention has broader applicability, including for such indications.

[0287] Other examples of diseases that can be treated with gene therapy using the secretory protein according to the invention include cardiovascular diseases and hematological disorders, especially coagulation disorders such as hemophilia (A, B or C), Winwib disease and factor VII deficiency.

[0288] Other examples of diseases or conditions requiring treatment 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 cytosolic storage diseases.

[0289] 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, retroviral / lentiviral (e.g., SIV) vector systems are 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 lung diseases 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 (compulsory pulmonary fibrosis) is provided.

[0290] This invention also provides a retroviral / lentiviral (e.g., SIV) vector as described herein, used in methods for treating diseases. Typically, the retroviral / lentiviral (e.g., SIV) vector system is produced using the methods of this invention. Any disease described herein can be treated according to this invention. In particular, this invention provides the retroviral / lentiviral (e.g., SIV) vector of this invention in a method for treating lung diseases. The disease to be treated may be a chronic disease. Preferably, a retroviral / lentiviral (e.g., SIV) vector for treating CF is provided.

[0291] This invention also provides the use of retroviral / lentiviral (e.g., SIV) vectors as described herein for manufacturing agents in methods for treating diseases. Typically, retroviral / lentiviral (e.g., SIV) vector systems are produced using the methods of this invention. Any disease described herein can be treated according to this invention. In particular, this invention provides the use of the retroviral / lentiviral (e.g., SIV) vectors of this invention for manufacturing agents in methods for treating lung diseases. The disease to be treated may be a chronic disease. Preferably, the invention provides the use of retroviral / lentiviral (e.g., SIV) vectors for manufacturing agents in methods for treating CF (compulsory pulmonary fibrosis).

[0292] Allocation and distribution The retroviral / lentiviral (e.g., SIV) vector of the present invention can be adapted to be administered at any dose to achieve the desired therapeutic effect. An appropriate dose can be determined by a clinician or other practicing physician using standard techniques and within the normal course of their practice. Non-limiting examples of suitable doses include 1 × 10⁻⁶. 8 One transduction unit (TU), 1×10 9 TU, 1×10 10 TU, 1×10 11 TU or more.

[0293] This invention also provides compositions 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. However, in some embodiments, the composition is in lyophilized form, in which case it may include a stabilizer, such as bovine serum albumin (BSA). In some embodiments, the composition may need to be formulated with a preservative (such as thimerosal or sodium azide) to facilitate long-term storage.

[0294] The retroviral / lentiviral (e.g., SIV) vectors of the present invention can be administered via any suitable route. It may be necessary to direct the compositions of the present invention (as described above) to the respiratory system of the subject. Effective delivery of therapeutic / prophylactic compositions or agents to the site of respiratory infection can be achieved via oral or nasal administration, for example, in the form of an aerosol (e.g., nasal spray) or via a catheter. Typically, the retroviral / lentiviral (e.g., SIV) vectors of the present invention are stable in clinically relevant nebulizers, inhalers (including pre-dose inhalers), catheters, and aerosols. Therefore, the retroviral / lentiviral (e.g., SIV) vectors of the present invention are typically formulated for administration to the lungs by any suitable means, such as endotracheal administration, intranasal administration, aerosol delivery, or direct injection or delivery to the lungs (e.g., via a catheter). The present invention also covers other delivery modalities, such as intravenous delivery.

[0295] In some embodiments, the nose is a preferred site for the production of 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) ease of vector administration; (iii) smaller vector quantities required; and (iv) ethical considerations. Therefore, transduction of nasal epithelial cells with the retroviral / lentiviral (e.g., SIV) vectors of the present invention can elicit effective (high-level) and durable expression of the therapeutic transgenic gene of interest. Therefore, nasal administration of the retroviral / lentiviral (e.g., SIV) vectors of the present invention is preferred.

[0296] The preparation for intranasal administration may be in the form of nasal drops or nasal spray. The intranasal preparation may contain droplets with a diameter generally in the range of 100-5000 µm, such as 500-4000 µm, 1000-3000 µm, or 100-1000 µm. Alternatively, in terms of volume, the droplets may be in the range of about 0.001-100 µl, such as 0.1-50 µl or 1.0-25 µl, or such as 0.001-1 µl.

[0297] Aerosol formulations can be in the form of powder, suspension, or solution. The size of the aerosol particles is related to the delivery capability of the aerosol. Smaller particles can travel further along the respiratory airway toward the alveoli compared to larger particles. In one embodiment, the aerosol particles have a diameter distribution that facilitates delivery along the entire length of the bronchi, bronchioles, and alveoli. Alternatively, the particle size distribution can be selected to target specific portions of the respiratory airway, such as the alveoli. In the case of aerosol-delivered drugs, the particle diameter can be in the general range of 0.1-50 µm, preferably 1-25 µm, more preferably 1-5 µm.

[0298] Aerosol particles can be delivered using a nebulizer (e.g., via the mouth) or a nasal spray. Aerosol formulations may optionally contain propellants and / or surfactants.

[0299] The formulation of pharmaceutical aerosols is routine for those skilled in the art; see, for example, Sciarra, J. in Remington's Pharmaceutical Sciences (ibid.). Pharmaceuticals can be formulated as dry powders, emulsions, or semi-solid formulations in the form of solution aerosols, dispersions, or suspensions. Aerosols can be delivered using any propellant system known to those skilled in the art. Aerosols can be applied, for example, by nasal inhalation to the upper respiratory tract, or to the lower respiratory tract, or both. The lung portion to which the pharmaceutical is delivered can be determined by the condition. Compositions containing the carrier of the present invention, especially when intended for intranasal delivery, may contain a humectant. This helps reduce or prevent mucosal dryness and irritation to the membrane. Suitable humectants include, for example, sorbitol, mineral oil, vegetable oil, and glycerin; soothing agents; membrane conditioners; sweeteners; and combinations thereof. Compositions may contain surfactants. Suitable surfactants include nonionic, anionic, and cationic surfactants. Examples of surfactants that can be used include, for example, polyoxyethylene derivatives of fatty acid esters of sorbitan, such as Tween 80, polyethylene glycol 40 stearate, polyoxyethylene 50 stearate, fusieate, bile salts, and octoxynol.

[0300] In some cases, subsequent administrations of the retroviral / lentiviral (e.g., SIV) vector may be performed after the initial administration. Administrations may be performed, for example, at least one week, two weeks, one month, two months, six months, one year, or longer after the initial administration. In some cases, the retroviral / lentiviral (e.g., SIV) vector 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, annually, or at longer intervals. Preferably, administration is performed every six months, more preferably annually. The retroviral / lentiviral (e.g., SIV) vector may be administered, for example, at intervals indicated by the decline in the effectiveness of previous administrations.

[0301] Any two or more retroviral / lentiviral (e.g., SIV) vectors of the present invention can be administered separately, sequentially, or simultaneously. Therefore, two 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) can be administered separately, simultaneously, or sequentially, and in particular, two or more retroviral / lentiviral (e.g., SIV) vectors of the present invention can be administered in this manner. Both can be administered in the same or different compositions. In a preferred embodiment, the two retroviral / lentiviral (e.g., SIV) vectors can be delivered in the same composition.

[0302] sequence homology A variety of sequence alignment methods can be used to determine the percentage of agreement, including but not limited to global methods, local methods, and hybrid methods such as segmentation methods. The procedure for determining the percentage of agreement is a standard procedure within the scope of those skilled in the art. Global methods align sequences from the start to the end of the molecule and determine the optimal alignment by accumulating scores for individual residue pairs and by imposing vacancy penalties. Non-restrictive methods include, for example, CLUSTAL W, see Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific GapPenalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see 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 input sequences.Non-restrictive methods include, for example, Match-box, see, for example, 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, for example, CE Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993); Align-M, see, for example, Ivo Van Walle et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004).

[0303] Therefore, the percentage of sequence identity is determined using conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986, and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. In short, the alignment of two amino acid sequences is optimized by using a 10-point vacancy opening penalty, a 1-point vacancy expansion penalty, and the Henikoff and Henikoff (ibid.) “blosum 62” scoring matrix as shown below (amino acids are indicated by standard single-letter codes).

[0304] The "percentage of sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of shared identical positions. Therefore, the identity percentage 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 the sequence identity percentage may also take into account the number and length of vacancies that need to be incorporated to optimize the alignment of the two or more sequences. Sequence comparisons and the determination of the identity percentage between two or more sequences can be performed using specific mathematical algorithms, such as BLAST, which will be familiar to those skilled in this technique.

[0305] Alignment score used to determine sequence consistency The percentage of consistency is calculated as follows: (Total number of consistent matches / length of the longer sequence plus the number of gaps introduced into the longer sequence for comparison) × 100 Essentially, homologous peptides are characterized by the presence of one or more amino acid substitutions, deletions, or additions. These changes are preferably minor, i.e., conserved amino acid substitutions (as described herein) and other substitutions that do not significantly affect the folding or activity of the peptide; small deletions, typically one to about 30 amino acids; and small amine or carboxyl-terminal extensions, such as amino-terminal methionine residues, small linker peptides of up to about 20-25 residues, or affinity tags.

[0306] In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovalinic acid, and α-methylserine) may replace amino acid residues in the polypeptides of the present invention. A limited number of non-conserved amino acids, amino acids not encoded by the genetic code, and non-natural amino acids may replace amino acid residues in the polypeptides. The polypeptides of the present invention may also contain non-naturally occurring amino acid residues.

[0307] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methyleneproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allethamine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamate, homoglutamate, 2-piperidinic acid, tertiary leucine, n-valine, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods for incorporating non-naturally occurring amino acid residues into proteins are known in this art. For example, an in vitro system can be used, in which chemically aminoacylated repressor tRNA is used to suppress nonsense mutations. Methods for synthesizing amino acids and aminoacylated tRNA are known in this art. Transcription and translation of plastids containing nonsense mutations were performed in a cell-free system containing *E. coli* S30 extract and commercially available enzymes and other reagents. Proteins were purified by chromatography. (See, for example, 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 a second method, translation was performed in *Xenopus* oocytes via microinjection of mutant mRNA and chemically aminoacylated repressor 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 desired natural amino acid (e.g., phenylalanine) and in the presence of the desired non-natural amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-natural amino acid is incorporated into the peptide to replace its natural counterpart. See Koide et al., *Biochem.* 33:7470-6, 1994. Naturally occurring amino acid residues can be converted into non-natural species through in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the substitution range (Wynn and Richards, *Protein Sci.* 2:395-403, 1993).

[0308] A limited number of non-conserved amino acids, amino acids not encoded by the genetic code, non-naturally occurring amino acids, and non-natural amino acids can replace the amino acid residues of the polypeptides of the present invention.

[0309] Essential amino acids in the peptides of this invention can be identified using procedures known in this 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 through physical structural analysis, using techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling combined with mutations in amino acids at hypothetical contact sites. See, for example, 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 properties of essential amino acids can also be inferred from homology analysis of related components (e.g., translocation or protease components) of the peptides of this invention.

[0310] Known methods of mutation induction and screening can be used to perform and test multiple amino acid substitutions, 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). In short, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting functional polypeptides, and then sequencing the mutation-induced polypeptide to determine the permissible substitution profile at each position. Other methods that may be used include phage presentation (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO92 / 06204) and region-specific mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0311] Example The invention is now described with reference to the following examples. These examples do not limit the scope of the invention, and those skilled in the art will understand that suitable equivalents can be used within the scope of the invention. Therefore, the examples can be considered as part of the invention, and the individual aspects described therein can be considered as disclosed independently or in any combination.

[0312] Example 1 - Modifying vector gene somatosomes, including reducing the number of intact SIV ORFs within the vector gene somatosome, maintaining or even increasing vector yield. The inventors reviewed the sequence of the constructed plasmid and identified several regions of interest within the original vector genomic plasmid pGM326. In particular, the Gag RRE cPPT hCEF region of pGM326 contains: ●77 start codons (ATG); ● 32 ORFs with a length of ≥ 10 amino acids ● Two large ORFs in the 5' to 3' direction ○The 189 amino acids starting from 5' ATG in the vector genome (Gag / RRE fusion) encode the p17 matrix and part of the p24 shell. ○ 250 amino acids starting from ATG inside RRE (RRE / cPPT / hCEF fusion) In particular, 14 ATG start codons were identified in the Gag / RRE region of the pGM326 gene ploid body, which produces an ORF longer than 10 amino acids. These start codons are plotted in... Figure 4 In the middle. The circled ATG is one with a strong kozak sequence and is related to... Gag or Env ATG in the same frame.

[0313] Therefore, the inventors designed a modified pGM326 plasmid with a combination of additional modifications aimed at reducing the number of intact SIV ORFs (and specifically removing these two large ORFs) to improve safety. Modifications were made to the hCEF promoter and the two large ORFs upstream of the CFTR transgene (soCFTR2). The changes are as follows: method One or more modifiers Editing area plastid 1 4 fsATGs Some Gags pGM826 2 2 fsATGs Some Gags pGM827 3 2 mtATGs Some Gags pGM828 4 mtSTOP + 1 mtATGs Some Gags pGM829 5 4 fsATGs + 3 mtATGs Partial Gag + RRE pGM830 6 mtSTOP + 4 mtATGs Partial Gag + RRE pGM831 fsATG = Frameshift ATG; mtATG = ATG with point mutations (broken ATG); mtSTOP = mutation ATG -> stop codon (introduction) Method 1 involves frameshift mutations of ATG codons (fsATG) 1, 2, 3, and 5 in the partial Gag region of the SIV-CFTR. Method 2 involves frameshift mutations of ATG codons 1 and 3 in the partial Gag region of the SIV-CFTR. Method 3 involves point mutations of ATG codons (mtATG) 1 and 3 in the partial Gag region of the SIV-CFTR. Method 4 mutates codon 6 in the partial Gag region of the SIV-CFTR into a stop codon and performs point mutations on ATG codon 3 in a partial Gag region. Method 5 involves frameshift mutations of ATG codons 1, 2, 3, and 5 in the partial Gag / RRE region of the SIV-CFTR and performs point mutations on ATG codons 7, 12, and 13. Method 6 mutates codon 6 in the partial Gag region of the SIV-CFTR into a stop codon and performs point mutations on ATG codons 3, 7, 12, and 13 across the partial Gag / RRE region of the SIV-CFTR. Method 5 produces... Figure 1A The pGM830 vector gene somatoform shown has the sequence SEQ ID NO: 19.

[0314] The functionality of novel vector genosomes was assessed through two rounds of transient lentiviral vector (LV) generation. The tested genosomes were transfected into A459 cells in 12 mL volumes using SIV GagPol, SIV Rev, SeV Fct4, ​​and SIVct+SeVHN genosomes within an Ambr® 15 bioreactor system. After LV generation, the vector products were activated, filtered through a 0.45 μm filter, and stored at -80°C. Upon thawing, the activated material was diluted 1:50 and transduced into A459 cells. The titer of the obtained LVs was quantified using CFTR FACS.

[0315] like Figure 5 As shown, several modified vector gene somatic ploids observably increased LV titer compared to the unmodified pGM326 vector gene somatic ploid. The increase was 1.0 × 10⁻⁶ compared to the unmodified pGM326. 6 Compared to TU / mL, the pGM830 vector gene plasmids produced the highest LV titer (6.5 × 10⁻⁶). 6 TU / mL).

[0316] In another identical production protocol, comparing vector titers using pGM326 and modified vector genosomes confirmed that using modified vector genosomes produced at least the same titer as pGM326, demonstrating that an improved safety profile could be achieved without negatively impacting titer.

[0317] Example 2 - Modifying vector gene somatosomes, including reducing the number of intact SIV ORFs within the vector gene somatosome, maintaining or even increasing vector integration. Example 1 LV generation was replicated using HEK239T cells.

[0318] The LV titer was quantified using a 3-day integration analysis. DNA was collected from transduced cells 3 days post-transduction, and unintegrated DNA was removed. Subsequently, qPCR was used to determine and quantify the presence / integration of vectors in the host cell DNA.

[0319] like Figure 6 As shown, compared with the unmodified pGM326 vector gene somatosomes, the modified pGM826 and pGM830 vector gene somatosomes observably increased LV integration. The increase was 9.3 × 10⁻⁶ compared to the unmodified pGM326. 5 Compared to TU / mL, the pGM830 vector gene somatosomes produced the highest LV integration (1.3 × 10⁻⁶). 6 TU / mL).

[0320] Similarly, in another identical production scheme, comparing vector titers using pGM326 and modified vector genosomes confirmed that using modified vector genosomes produced at least LV integration equivalent to pGM326, demonstrating that an improved safety profile can be achieved without adversely affecting LV functionality.

[0321] Example 3 - Modifying vector gene ploids, including reducing the number of intact SIV ORFs within the vector gene ploid, maintaining or even increasing transgenic gene 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 post-transduction, and subsequently, cells transduced only in the absence of inhibitors were passaged and stained again for CFTR expression 10 days post-transduction to investigate the extent of pseudotransduction (transduction that does not integrate proviral DNA into the host genome), which can also induce CFTR expression.

[0322] like Figure 7 As shown, when using the reverse transcription inhibitor (azidothymidine AZT) and the SIV integration inhibitor (ralteravir), the number of cells exhibiting CFTR was almost the same as that of the negative control, indicating that CFTR is a result of LV integration.

[0323] also, Figure 7 It was also shown that, compared with LVs generated using pGM326, even when AZT was included during transduction, LVs generated using pGM830 had a higher percentage of CFTR-positive cells.

[0324] Therefore, this comparison of CFTR transgenic expression using pGM326 and pGM830 confirms that using modified vector genotypes produces at least comparable transgenic expression compared to LVs produced using unmodified pGM326, indicating that an improved safety profile can be achieved without adversely affecting LV functionality.

[0325] Example 4 - Cleavage of Fct4 with an enzyme having trypsin-like cleavage specificity to produce a fusion-active form containing F1 and F2 fragments. After the addition of a trypsin-like enzyme, the cleavage of the F protein in the LV produced according to Example 1 was evaluated. Activation of the F protein occurs through cleavage into two subunits, F1 and F2. Therefore, the cleavage of the F protein is the accepted substitute for F protein activation and thus fusion capability.

[0326] After incubating LV with trypsin-like enzyme, the Western ink dot method was performed using anti-PIV1 antibody ab20791 at a 1:5000 dilution. Figure 8 As shown, Fct4 was successfully cleaved when cultured with a trypsin-like enzyme. In the presence of this enzyme, uncleaved F0 was not detected; only F1 was detected.

[0327] Sequence information Sequence retrieval table 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 SEQ ID NO: 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 Figure 1A The plastid (pDNA1 pGM830) defined in [the text]. SEQ ID NO:20 Figure 1B The plastid (pDNA1 pGM691) defined in [the text]. SEQ ID NO: 21 Figure 1C The plastid (pDNA2a pGM297) defined in [the text]. SEQ ID NO: 22 Figure 1D The plastid (pDNA2b pGM299) defined in [the text]. SEQ ID NO:23 Figure 1E The plastid (pDNA3a pGM301) defined in [the text]. SEQ ID NO: 24 Figure 1F The plastid (pDNA3b pGM303) defined in [the text]. SEQ ID NO: 25 Figure 1G The plastid (pDNA2a pGM326) as defined in [the document / reference]. 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 Complementary strand of exemplary A1AT transgene SEQ ID NO: 32 Exemplary A1A1 peptide SEQ ID NO: 33 Exemplary FVIII transgene (N6) SEQ ID NO: 34 Exemplary FVIII transgene (V3) SEQ ID NO: 35 Complementary strand of exemplary FVIII transgene (N6) SEQ ID NO: 36 Complementary strand of exemplary FVIII transgene (V3) SEQ ID NO: 37 Exemplary FVIII peptide (N6) SEQ ID NO: 38 Exemplary FVIII peptide (V3) SEQ ID NO: 39 Exemplary WPRE component (mWPRE) SEQ ID NO: 40 Figure 2 The F / HN-SIV-hCEF-soA1AT plasmid (pDNA1 pGM407) is defined in [the text]. SEQ ID NO: 41 Figure 3A The F / HN-SIV-CMV-HFVIII-V3 plastid (pDNA1 pGM411) defined in [the text]. SEQ ID NO: 42 Figure 3B The F / HN-SIV-hCEF-HFVIII-V3 plastid (pDNA1pGM413) defined in [the text]. SEQ ID NO: 43 Figure 3C The F / HN-SIV-CMV-HFVIII-N6-co plasmid (pDNA1pGM412) is defined in [the text]. SEQ ID NO: 44 Figure 3D The F / HN-SIV-hCEF-HFVIII-N6-co plasmid (pDNA1pGM414) is defined in [the text]. SEQ ID NO: 45 Exemplary CAG Promoter SEQ ID NO: 46 Additional amino acid sequence encoded by the pseudo-transcription start site upstream of the Fct4 amino acid sequence of SEQ ID NO: 13. sequence <210> SEQ ID NO: 1 <211> 7553 <223> Modified SIV / CFTR RNA sequence ucucuuacua ggagaccagc uugagccugg guguucgcug guuagccuaa ccugguuggc 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 ugguauuu augguaacug cccacuuggc aguacaucaa guguaucaua 1920 ugccaaguau gcccccuauu gaugucaaug augguaaaug gccugccugg cauuaugccc 1980 aguacaugac cuuaugggac uuuccuacuu gccaguacau 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 aguggaggac 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 cuucgcacuu uuuaaaagaa 7380 aagggaggac uggaugggau uuauuacucc gauaggacgc uggcuuguaa cucagucucu 7440 uacuaggaga ccagcuugag ccuggguguu cgcugguuag ccuaaccugg uuggccacca 7500 gggguaagga cuccuuggcu 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 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 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 Val Cys Val Leu Tyr Cys Leu His Lys Glu Gln Lys Val 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 Leo Val Val Ile Ile Val Ile Ile Ile Val 485 490 495 Leo Tyr Arg Leo 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 Leu 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(fragment2) 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 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 cggcaaagag 120 atggaaagat tcggcctgca cgagcggctg ctggaaaccg aggaaggctg caagagaatt 180 atcgaggtgc tgtaccctct ggaacctacc ggctctgagg gcctgaagtc cctgttcaat 240 ctcgtgtgcg tgctgtactg cctgcacaaa gaacagaaag tgaaggacac cgaagaggcc 300 gtggccacag ttagacagca ctgccacctg gtggaaaaag agaagtccgc cacagagaca 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 gacactgag agatcctaga 720 ggctctgata tcgccggcac caccagctct gtgcaagagc agctgggaatg gatctacacc 780 gccaatccta gagtggacgt gggcgccatc tacaagaat 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 ggcctcctctgagatgctac 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 ggcgctagag gaccttgtgt gcgacagtgg cctctgagca 1920 aagagaagat tgaggccctg caagaaatct gtagccagct ggaacaagag ggcaagatca 1980 gcagagttgg cggcgagaac gcctacaata cccctatctt ctgcatcaag aaaaaggaca 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 tggctcaca agtgggagct gagccggatt cagctcgaag 2580 agaaggacga gtggaccgtg aacgacatcc agaactcgt gggcaagctg aattgggcag 2640 cccagctgta tcccggcctg aggaccaga acatctgcaa gctgatccgg ggaaagaga 2700 acctgctgga actgtcaca 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 ggaaagatt gaagaggccc 3540 agaggaaca cgagcgctac cacaact ggaagaatct ggccgacacc tacggactgc 3600 cccagatcgt ggccaaagaa atcgtggcta tgtgccccaa gtgtcagatc aagggcgaac 3660 ctgtgcacgg ccaagtggat gcttctcctg gcacatggca gatggactgt accacctgg 3720 aaggcaaagt gtcatcgtg gctgtgcacg tggcctccgg ctttattgag gccgaagtga 3780 tccccagaga gataggcaa gaaccgcca agttcctgct gaagagatcctg tccagatggc 3840 ccatcacaca gctgcacacc wocacggcc ctactcac atctcaagag gtggccgcca 3900 tctgttggtg gggaaagatt gagcacacaa ccggcattcc ctacaatcca cagagccagg 3960 gcagcatcga gtccatgaac aagcagctca aagagatt cggcagatc cgggacgact 4020 gccagtacac agaacagcc gtgctgatgg cctgtcacat ccacactc aagcggaaag 4080 gcggcatcgg aggacaca tctgccgaga gactgatca tatcatcacc actcagctgg 4140 aaatccagca cctccagacc aagatccaga agattctgaa cttccgggtg tactaccgcg 4200 agggcagaga tcctgttgg aaggcccag cacagctgat ctggaaggc gaaggtgccg 4260 tggtgctgaa ggatggctct gatctgaagg tggtgcccag acggaggcc aagatttaca 4320 aggattacga gcccaacag cgcgtggggca atgaggcga cgttgagggc acagaggca 4380 gcgacaattg a 4391 <210> SEQ ID NO: 18 <211> 4391 <213> Beautiful sweets and sweets in gagpol atggggggcgg ctacctcagc actaaatagg agacaattag accaatttga gaaaatacga 60 cttcgcccga acggaagaa aaagtaccaaattaacatt taatatggcc agggaaggag 120 atggagcgct tcggcctcca tgagaggttg tggagacag aggaggggtg tAAagaatc 180 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 aacaacaagg aaatgcctgg gtacatgtac ccttgtcacc gcgcacctta 480 aatgcgtggg taaaagcagt agaggagaaa aaatttggag cagaaatagt acccatgttt 540 caagccctat cagaaggctg cacaccctat gacattaatc agatgcttaa tgtgctagga 600 gatcatcaag gggcattaca aatagtgaaa gagatcatta atgaagaagc agcccagtgg 660 gatgtaacac acccactacc cgcaggaccc ctaccagcag gacagctcag ggaccctcgc 720 ggctcagata tagcagggac caccagctca gtacaagaac agttagaatg gatctatact 780 gctaaccccc gggtagatgt aggtgccatc taccggagat ggattattct aggacttcaa 840 aagtgtgtca aaatgtacaa cccagtatca gtcctagaca ttaggcaggg acctaaagag 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 aaatcaaaca tggtgcagca gggaggtcca aaaagacaaa gaccccact aagatgttat 1200 aattgtggaa aatttggcca tatgcaaga caatgtccgg aaccaaggaa aacaaatgt 1260 ctaaagtgtg gaaaattggg acacctagca aaagactgca ggggacaggt gatttttta 1320 gggtatggac ggtggatggg ggcaaaccg agaatttc ccgccgctac tcttggagcg 1380 gaaccgagtg cgcctccctc accgagcggc accacccat acgacccagc aagaagctc 1440 ctgcagcaat aggcagagaa agggaaaaaaaaaaggaggaa tccaccggca 1500 atgaatccgg attggaccga gggatattct tgaacccc tctttggaga agaccataa 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 gatcataga aagcatgac aacattaa 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 cgtggaggt accagggat 4380 ctgataacta a 4391 <210> SEQ ID NO: 19 <211> 10536 <223> pGM830 ggtacctcaa tattggccat tagccatatt attcattggt tatatagcat aaatcaat 60 tggctattgg ccattgcata cgttgtatct atatcataat atgtacatttt atattggctc 120 atgtccaata tgaccgccat gttggcattg attattgact agttattaat agtaatcaat 180 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 240 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 300 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 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 tctgacccaggcgagagaaa 960 1020 ggcgtcggac gcgaaggaag cgcggggtgc gacgcgacca agaggagac 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 acagagggg ggtgtaaaag aatcatagaa gtcctctacc 1380 ccctagaacc aacaggatcg gagggcttaa aaagtctgtt caatcttgtg tgcgtgctat 1440 attgcttgca caaggaacag aaagtgaaag acacagaga agcagtagca acagtaagac 1500 aacactgcca tctagtggaa aaagaaaaaa gtgcaacaga gacatctagt ggacaaaaga 1560 aaaatgacaa gggaatagca gcgccacctg gtggcagtca gaatttcca gcgcaacaac 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 gactaattaa tataataaca acacaattag aaatacaaca tttacaaacc 2460 aaaattcaaa aaattttaaa ttttagagcc gcggagatct gttacataac ttatggtaaa 2520 tggcctgcct ggctgactgc ccaatgaccc ctgcccaatg atgtcaataa tgatgtatgt 2580 tcccatgtaa tgccaatagg gactttccat tgatgtcaat gggtggagta tttatggtaa 2640 ctgcccactt ggcagtacat caagtgtatc atatgccaag tatgccccct attgatgtca 2700 atgatggtaa atggcctgcc tggcattatg cccagtacat gaccttatgg gactttccta 2760 cttggcagta catctatgta ttagtcattg ctattaccat gggaattcac tagtggagagaa 2820 gagcatgctt gagggctgag tgcccctcag tgggcagaga gcacatggcc cacagtccct 2880 gagaagttgg ggggaggggt gggcaattga actggtgcct agaaggtg gggcttgggt 2940 aaactgggaa agtgatgtgg tgtactggct ccaccttttt ccccagggtg ggggagaacc 3000 atatataagt gcagtagtct ctgtgaacat tcaagcttct gccttctccc tcctgtgagt 3060 ttgctagcca ccatgcagag aagccctctg gagaaggcct ctgtggtgag caagctgttc 3120 ttcagctgga ccaggcccat cctgaggaag ggctacaggc agagactgga gctgtctgac 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 3660 ctggcccact ttgtgtggat tgcccctctg caggtggccc tgctgatggg cctgatttgg 3720 gagctgctgc aggcctctgc cttttgtggc ctgggcttcc tgattgtgct ggccctgttt 3780 caggctggcc tgggcaggat gatgatgaag tacagggacc agaggcagg caagatcagt 3840 gagaggctgg tgatcacctc tgagatgatt gagacatcc agtctgtgaa ggcctactgt tgggaggag ctatggaga gatgattga aacctgaggc agacagagct gagctgacc aggaaggctg cctatgtgag atacttcaac agctctgcct tcttcttctc tggcttcttt gtggtgttcc tgtctgtgct gccctatgcc ctgatcaagg ggatcatcct gagaagatt 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 4380. aacaacagaa agaccagcaa tggggatgac tccctgttct tctccaactt ctccctgctg 4440. ggcacacctg tgctgaagga catcaacttc aagattgaga gggggcagct gctggctgtg gctggatcta caggggctgg caagaccagc ctgctgatga tgatcatggg ggagctggag ccttctgagg gcaagatcaa gcactctggc aggatcagct tttgcagcca gttcagctgg atcatgcctg gcaccatcaa ggagaacatc atctttggag tgagctatga tgagtacaga 4620 tacaggagtg tgatcaggc ctgccagctg gaggaggaca tcagcagtt tgctgagag 4680 gandacattg tgctggggga gggaggcatt acactctg ggggccagag agccagaatc 4740 agcctggcca gggctgtgta caagctgct gacctgtacc tgctggactc cccctttggc 4800 tacctggatg tgctgacaga gaggagat tttgagagct gtgtgtgcaa gctgatggcc 4860 aacaagacca gatcctggt gaccagcaag atggagcacc tgagaaggc tgacaagatc 4920 ctgatcctgc atgagggcag cagctacttc tatgggacct tctctgagct gcagaacctg 4980 cagcctgact tcagctctaa gctgatggggc tgtgacagct tgaccagtt ctctgctgag 5040 aggaggaaca gcatcctgac agagaccctg cacagattca gcctggaggg agatgcccct 5100 gtgagctgga cagagaccaa gaagcagagc ttcaagcaga caggggagtt tgggagag 5160 aggaagaact ccatcctgaa cccatcaac agcatcagga agttcagcat tgtgcagaaa 5220 acccccctgc agatgaatgg cattgaggaa gattctgatg agcccctgga gaggagactg 5280 agcctggtgc ctgattctga gcagggagag gccatcctgc ctaggatctc tgtgatcagc 5340 acaggcccta cactgcaggc cagaaggagg cagtctgtgc tgaacctgat gacccactct 5400 gtgaaccagg gccagaacat ccacaggaaa accacagcct ccaccaggaa agtgagcctg 5460 gcccctcagg ccaatctgac agagctggac atctacagca ggaggctgtc tcaggagaca 5520 ggcctggaga tttctgagga gatcaatgag gaggacctga aagagtgctt ctttgatgac 5580 atggagagca tccctgctgt gaccacctgg aacacctacc tgagatacat cacagtgcac 5640 aagagcctga tctttgtgct gatctggtgc ctggtgatct tcctggctga agtggctgcc 5700 tctctggtgg tgctgtggct gctgggaaac accccactgc aggacaaggg caacagcacc 5760 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 acaagcctga agggcctgtg gaccctgaga gccttttggca ggcagcccta ctttgagacc 6300. ctgttccaca aggccctgaa cctgcacaca gccaactggt tcctctacct gtccaccctg agatggttcc agatgagat tgatgatc 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 ctggagaaca tctccttcag catctcccct 6780 ggccagagag tgggactgct gggaagaaca ggctctggca agtctaccct gctgtctgcc 6840 ttcctgaggc tgctgaacac agagggagag atccagattg atggagtgtc ctgggacagc 6900 atcacactgc agcagtggag gaaggccttt ggtgtgatcc cccagaaagt gttcatcttc 6960 agtggcacct tcaggaagaa cctggacccc tatgagcagt ggtctgacca ggagatttgg 7020 aaagtggctg atgaagtggg cctgagaagt gtgattgagc agttccctgg caagctggac 7080 tttgtcctgg tggatgggg ctgtgtgctg agccatggcc acaagcagct gatgtgcctg 7140 gccagatcag tgctgagcaa ggccaagatc 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 gctgggacatc agacagggac ccaaagagcc 2640 cttcaaggac tacgtggacc ggttctataa ggccattaga gccgagcagg ccagcgggga 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 gagagagagg cctcctctga gatgctacaa 2940 ctgcggcaag ttcggccaca tgcagagaca gtgtcctgag cctaggaaaa caaaatgtct 3000 aaaggtgga aaattgggac acctagcaaa agactgcagg ggacaggtga attttttagg 3060 gtatggacgg tggatgggg caaaaccgag aaattttccc gccgctactc ttggagcgga 3120 accgagtgcg cctctcccac cgagcggcac cacccatac gacccagcaa agaagctcct 3180 gcagcaatat gcagagaaag ggaaacaact gagggagcaa aaggaatc caccggcaat 3240 3300 accgtgtaca tcgagggcgt gcccatcaag gctctgctgg atacaggcgc cgacgacacc 3360 atcatcaaag agaacgacct gcagctgagc ggcccttgga ggcctaagat cattggagga 3420 atcggcggag gcctgaacgt caaagagtac aacgaccggg aagtgaagat cgaggacaag 3480 atcctgaggg gcacaatcct gctgggcgcc acacctatca acatcatcgg cagaaatctg 3540 ctggcccctg ccggcgctag actggttatg ggacagctct ctgagaagat ccccgtgaca 3600 cccgtgaagc tgaaagaagg cgctagagga ccttgtgtgc gacagtggcc tctgagcaaa 3660 gagaagattg 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 gccttcacaa tccccaccgt gaacaatcaa 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 tggaaggcgg ccagtggtcc taccagttta agcaagaagg ccaggtcctg 4620 aaagtgggca agtacaccaa gcagagaac acccacacca acgagctgag gacactggct 4680 ggcctggtcc agaaaatctg caaagaggcc ctggtcattt ggggcatcct gcctgttctg 4740 gaactgccca ttgagcggga agtgtgggaa cagtggtggg ccgattactg gcaagtgtct 4800 tggatccccg agtgggactt cgtctacc cctcctctgc tgaactgtg gtacaccctg 4860 aaaagagc ccattcctaa agaggacgtc tactacgttg acggcgcctg siaccggaac 4920 tccaaagaag gcaaggccgg ctacatcagc cagtacggca agcagagagt ggaaccctg 4980 gaaaacacca ccaccagca ggccgagctg accgccatta agatggccct ggaagatagc 5040 ggccccaatg tgaacatcgt gaccgactct cagtacgcca tgggaatcct gagcccag 5100 cctacacaga gcgatagccc tctgttgag cagatcattg ccctgatt tcagaagcag 5160 ...

Claims

1. A retroviral vector comprising a modified retroviral RNA sequence, characterized in that: The modified retroviral RNA sequence is compared to the unmodified retroviral RNA sequence from which the modified retroviral RNA sequence is derived. (i) by codon substitution; and (ii) contain a reduced number of retroviral open reading frames (ORFs); And among them: (a) the retroviral RNA sequence comprises a promoter and a transgene; and (b) The retroviral vector was pseudopatterned with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxovirus.

2. The retroviral vector according to claim 1, characterized in that Compared to the unmodified retroviral RNA sequence from which the modified retroviral RNA sequence is derived, the modified retroviral RNA sequence lacks: (a) one or more retroviral ORFs 5' to the promoter; (b) one or more retroviral ORFs encoding a peptide of ≥100 amino acids in length; (c) Included in part RRE One or more retroviral ORFs in the sequence; and / or (d) Included in part Gag One or more retroviral ORFs in the sequence.

3. The retroviral vector according to claim 1 or 2, characterized in that The respiratory paramyxovirus is Sendai virus.

4. The retroviral vector according to any one of the preceding claims, characterized in that 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; optionally wherein the vector comprises a hybrid human CMV enhancer / EF1a (hCEF) promoter.

5. The retroviral vector according to any one of the preceding claims, characterized in that The transgenic gene is selected from: a) CFTR , ABCA3 , DNAH5 , DNAH11 , DNAI1 and DNAI2 ;or b) Secretory 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.

6. The retroviral vector according to any one of the preceding claims, characterized in that The transgenic gene encodes: a) CFTR; b) A1AT; or c) FVIII.

7. The retroviral vector according to any one of the preceding claims, characterized in that: a) the promoter is the hCEF promoter and the transgenic gene encodes CFTR; b) the promoter is the hCEF promoter and the transgenic gene encodes A1AT; or c) The promoter is hCEF or CMV promoter and the transgenic gene encodes FVIII.

8. The retroviral vector according to any one of the preceding claims, characterized in that It is a lentiviral vector; optionally wherein 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 the preceding claims, characterized in that The retroviral vector is a SIV vector.

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

1.

11. The retroviral vector according to claim 10, characterized in that The modified retroviral RNA sequence comprises or consists 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.

12. The retroviral vector according to any one of the preceding claims, characterized in that The vector further comprises one or more of the following: (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 at most 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 at most 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 at most 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 at most 100% sequence identity to SEQ ID NO: 8; Optionally, the vector comprises each of (a) to (g).

13. A retroviral vector according to any one of the preceding claims, characterized in that The vector further comprises one or more of the following: (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 at most 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.

14. A retroviral vector according to any one of the preceding claims, characterized in that The F protein is a Fct4 protein, which optionally: (a) 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 at most 100% sequence identity to SEQ ID NO: 12 or 13; and / or (b) cleaved into two subunits, wherein optionally, 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.

15. A SIV vector, characterized in that Pseudopatterned with Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) proteins, where: (a) the vector comprises a modified retroviral RNA sequence comprising or consisting 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; 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.

16. The SIV vector according to claim 15, wherein The vector further comprises one or more of the following: (a) p17 protein, which comprises or consists 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).

17. A method for producing a retroviral vector as defined in any one of the preceding claims, characterized in that The method comprises the following steps: a) Suspension-growing cells; b) transfecting the cell with one or more plasmids; c) adding nuclease; d) Collecting lentivirus; e) adding trypsin or an enzyme with equivalent cleavage specificity; and f) Purification.

18. The method according to claim 17, characterized in that Steps (a) to (f) are performed sequentially.

19. The method according to claim 17 or 18, characterized in that The cells are HEK293T or 293T / 17 cells.

20. The method according to any one of claims 17 to 19, characterized in that Nuclease addition is performed at the pre-collection stage.

21. The method according to any one of claims 17 to 20, characterized in that The addition of trypsin or an enzyme with the same cleavage specificity is performed at a post-harvest stage.

22. The method according to any one of claims 17 to 21, characterized in that The purification step comprises a chromatography step.

23. A composition, characterized in that Comprising the retroviral vector according to any one of claims 1 to 16 and a pharmaceutically acceptable excipient or diluent.

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

25. Use of a retroviral vector as claimed in any one of claims 1 to 16 in a method of treatment.

26. A method for treating a disease, characterized in that: The method comprises administering the retroviral vector according to any one of claims 1 to 16 to a subject in need thereof.

27. Use of a retroviral vector according to claim 25 in a therapeutic method or a therapeutic method according to claim 26, characterized in that: The disease to be treated is a lung disease, preferably cystic fibrosis.

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