Gene therapy

Abstract

The present invention relates to gene therapy agents for the treatment of pulmonary alveolar proteinosis (PAP), particularly autoimmune PAP (aPAP). Specifically, the present invention relates to gene therapy vectors that drive transient and / or low-level expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), resulting in a therapeutic effect without the toxicity associated with the therapy. The present invention further relates to related products and animal models of aPAP.

Description

[Technical Field]

[0001] The present invention relates to gene therapy agents for the treatment of pulmonary alveolar proteinosis (PAP), particularly autoimmune PAP (aPAP). Specifically, the present invention relates to gene therapy vectors that drive transient and / or low-level expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), resulting in a therapeutic effect without the toxicity associated with the therapy. The present invention further relates to related products and animal models of aPAP. [Background technology]

[0002] Pulmonary alveolar proteinosis (PAP) is a rare autoimmune lung disease with currently inadequate treatment options and no pharmacological treatments approved for clinical use. Most PAP cases are caused by the presence of anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibodies that block surfactant clearance by alveolar macrophages. The current standard treatment for aPAP is whole lung lavage (WLL), a procedure in which lipoprotein surfactant is sequentially washed from each lung under anesthesia. WLL is undesirable for several reasons: it is invasive; it must be performed in a specialized center; it only treats symptoms; and it can cause complications. Furthermore, approximately 20% of patients require multiple interventions.

[0003] Currently, there are no approved pharmacological treatments for aPAP. However, recombinant GM-CSF protein has been administered to patients subcutaneously or by aerosol to overcome anti-GM-CSF antibodies and restore surfactant clearance. Meta-analyses of these case studies suggest that GM-CSF therapy for aPAP may be effective and that administration by the inhaled route appears superior to subcutaneous injection. A recent double-blind, placebo-controlled trial confirmed that daily administration of inhaled GM-CSF resulted in modest improvements in pulmonary gas exchange and functional health compared with placebo.

[0004] Gene therapy offers several advantages over recombinant protein-based therapies, namely, less frequent dosing requirements and more stable steady-state concentrations of therapeutic proteins, which may further enhance the therapeutic index. The UKCF Gene Therapy Consortium has previously generated lentiviral vectors pseudotyped with the F / HN protein from Sendai virus (rSIV.F / HN), specifically designed to achieve highly efficient lung targeting.

[0005] However, a common problem in gene therapy is the difficulty of producing enough protein to reach the therapeutic threshold required to treat or cure a disease. Therefore, achieving sufficient gene expression is a major obstacle to the success of many gene therapies, and existing treatments require administering large amounts of gene therapy agents to patients, exceeding 1 trillion viruses per kg of body weight. For example, Zolgensma delivers 1.1 x 10 per kg of body weight. 14A single viral genome is administered. Producing such large amounts of virus is expensive, contributing to gene therapy costs of $1,000,000, and administering such large amounts to humans could trigger an immune response that threatens the patient's health and the efficacy of the therapy. To circumvent these issues, research to date has focused on gain-of-function mutations that result in more potent proteins. Such approaches have previously been used in gene therapy for hemophilia B (the Padua mutation in factor IX) and lipoprotein lipase deficiency (the S447X variant of lipoprotein lipase).

[0006] However, because GM-CSF has a narrow therapeutic window, such conventional approaches and agents are not always applicable to aPAP.

[0007] Thus, there is an unmet clinical need for new technologies for the successful treatment of aPAP. It is an object of the present invention to address one or more of these problems. Specifically, it is an object of the present invention to provide novel gene therapy vectors that drive transient and / or low-level expression of GM-CSF, resulting in a therapeutic effect without the toxicity associated with such therapy. Summary of the Invention [Problem to be solved by the invention]

[0008] Currently, there remains an urgent need for technology that enables gene therapy that can provide GM-CSF in a tightly controlled manner so that GM-CSF is produced at concentrations within a narrow therapeutic window. The present inventors have demonstrated for the first time that transient, low-level GM-CSF expression can be achieved using regulated GM-CSF expression using viral or non-viral vector systems. Furthermore, the present inventors have surprisingly shown that such transient and / or low-level GM-CSF expression can alleviate the PAP phenotype in a mouse model of aPAP, allowing GM-CSF expression to be stopped at a time point before toxicity has been reported in the art.

[0009] Accordingly, the present invention provides a granulocyte-macrophage colony-stimulating factor (GM-CSF) gene therapy agent for use in the treatment of pulmonary alveolar proteinosis (PAP), which agent transiently expresses GM-CSF in a patient. [Means for solving the problem]

[0010] The transient GM-CSF protein expression can be 6 months or less, preferably 4 months or less, and more preferably 3 months or less. The treatment can reduce one or more PAP biomarkers selected from (a) bronchoalveolar lavage fluid (BALF) turbidity; (b) surfactant protein D (SF-D) concentration in the lung; (c) SF-D concentration in the BALF; (d) surfactant deposition in the lung; and / or (e) optionally a pulmonary pathology selected from (i) lung opacity, (ii) pulmonary edema, and / or (iii) lung consolidation. Alternatively, or in addition, the treatment may increase pulmonary function, optionally selected from increasing (i) vital capacity (VC); (ii) forced vital capacity (FVC); and / or (iii) forced expiratory volume (FEV), particularly FEV1; (iv) arterial oxygen tension (Pa,O2); (v) alveolar-arterial oxygen gradient (PA-a,O2); (vi) peak metabolic equivalent (peak METS), and / or (vii) six-minute walking distance (6MWD), preferably PA-a,O2.

[0011] The treatment may not be associated with one or more histopathological changes in the patient, the one or more histopathological changes optionally including (a) one or more histopathological changes in the lung, optionally including distortion of lung architecture, pulmonary inflammatory cell infiltration exceeding the PAP phenotype, increased alveolar wall thickening, pulmonary alveolar microlithiasis (PAM) alveoli, PAM bronchi, presence of neutrophils in the bronchi, consolidation, presence of giant cells, eosinophilic material, and / or edema; (b) one or more histopathological changes in the liver, optionally including distortion of lung architecture, pulmonary inflammatory cell infiltration exceeding the PAP phenotype, increased alveolar wall thickening, pulmonary alveolar microlithiasis (PAM) alveoli, PAM bronchi, presence of neutrophils in the bronchi, consolidation, presence of giant cells, eosinophilic material, and / or edema; (c) one or more histopathological changes in the kidney, optionally inflammatory cell infiltration exceeding the PAP phenotype, vascular dilation, fibrosis, eosinophilic material, and / or cysts; and / or (d) one or more histopathological changes in the spleen, optionally the presence of megakaryocyte clusters and / or macrophages.

[0012] The agent may comprise (a) a non-viral nucleic acid molecule encoding GM-CSF and a lipid carrier; or (b) a viral vector or non-viral nucleic acid molecule comprising a GM-CSF transgene operably linked to an inducible promoter.

[0013] The non-viral nucleic acid molecule can be a plasmid containing a GM-CSF transgene operably linked to a promoter. The GM-CSF transgene; the promoter; or both the GM-CSF transgene and the promoter can each contain 10 or fewer CpG dinucleotides, or no CpG dinucleotides. The plasmid can contain a GM-CSF transgene operably linked to a promoter 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, the plasmid contains a hybrid human CMV enhancer / EF1a (hCEF) promoter. The non-viral nucleic acid molecule can be an mRNA or self-amplifying RNA (saRNA) encoding GM-CSF. The mRNA may contain pseudouridine (ψ-UTP), Cap1, and / or a poly(A) tail of about 10-100 adenosine nucleotides, and optionally the mRNA is about 0.5 kb to about 5 kb in length. The saRNA may contain ψ-UTP, Cap1, and / or a poly(A) tail of about 10-100 adenosine nucleotides, and optionally the saRNA is about 9 kb to about 12 kb in length.

[0014] The lipid carrier may (i) be a lipid nanoparticle, preferably a liposome; (b) comprise one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids; and / or (c) be GL67A.

[0015] The agent may be a viral vector that is a lentiviral or retroviral vector. The lentiviral or retroviral vector may be (a) pseudotyped with (i) the hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus, preferably Sendai virus, or (ii) the G glycoprotein from vesicular stomatitis virus (G-VSV); and / or (b) a lentiviral vector, preferably an SIV vector, selected from the group consisting of simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV) vector, feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), and Visna / Maedi virus vector.

[0016] The inducible promoter can be (i) a steroid-regulated promoter, preferably a mifepristone-regulated promoter; or (ii) a chemically-regulated promoter. Alternatively, or in addition, (i) the transgene operably linked to the inducible promoter and the transactivator of the inducible promoter are contained in (i) the same lentiviral or retroviral vector, or (ii) separate lentiviral or retroviral vectors.

[0017] Medicaments for use in the present invention are formulated for pulmonary administration; optionally, administration is by intratracheal or intranasal instillation, aerosol delivery, nebulization, intravenous injection, or direct injection into the lungs.

[0018] The medicament may be for use in the treatment of autoimmune PAP (aPAP).

[0019] The present invention also provides a method for treating PAP, comprising administering a therapeutically effective amount of a GM-CSF gene therapy agent to a patient in need thereof.

[0020] The present invention further provides the use of a GM-CSF gene therapy agent in the manufacture of a medicament for the treatment of PAP.

[0021] The present invention also provides a composition comprising (a) a non-viral nucleic acid molecule encoding GM-CSF and a lipid carrier; or (b) a viral vector comprising a GM-CSF transgene operably linked to an inducible promoter, wherein the composition is formulated for administration to the lung, so that when administered, the non-viral nucleic acid molecule or viral vector can transiently express GM-CSF in lung cells.The non-viral vector can be a plasmid as defined herein; or the non-viral vector can be an mRNA or saRNA as defined herein; preferably, the lipid carrier can be as defined herein.

[0022] The present invention also provides a rodent model of aPAP, wherein the rodent is passively immunized with an anti-GM-CSF antibody by intranasal administration, where: (a) the rodent can be a mouse, optionally a mouse having a C57 Black 6 background, a wild-type mouse, or a GM-CSF knockout mouse; (b) the anti-GM-CSF antibody can be a mouse anti-GM-CSF antibody; and / or (c) the model can achieve a BALF anti-GM-CSF antibody concentration of about 4-6 μg / mL or more.

[0023] The present invention also provides a method for generating a rodent model of aPAP, comprising administering an anti-GM-CSF antibody to the rodent via intranasal administration, wherein: (a) the rodent can be a mouse, optionally a mouse having a C57 Black 6 background, a wild-type mouse, or a GM-CSF knockout mouse; and / or (b) the anti-GM-CSF antibody can be a mouse anti-GM-CSF antibody; and / or (c) the model can achieve a BALF anti-GM-CSF antibody concentration of about 4-6 μg / mL or greater.

[0024] The present invention also provides the use of a rodent model as defined herein (a) to study aPAP; and / or (b) to study drugs, cell products, biopharmaceuticals, or small molecules intended for the treatment of aPAP, and optionally to study compositions as defined herein. [Brief explanation of the drawings]

[0025] [Figure 1A] (a) Schematic linear representation of the pIC017 hCEF GMCSF plasmid, containing the mGM-CSF transgene under the control of the hCEF promoter, a bovine growth hormone (BGM) polyA sequence, an R6K origin of replication (containing CpG dinucleotides), and a kanamycin resistance cassette (also containing CpG dinucleotides). (b) Schematic linear representation of the pIC098 CMV GMCSF plasmid, containing the mGM-CSF transgene under the control of the CMV promoter, a bovine growth hormone (BGM) polyA sequence, an R6K origin of replication (containing CpG dinucleotides), and a kanamycin resistance cassette (also containing CpG dinucleotides). [Figure 1B] (a) Schematic linear representation of the pIC017 hCEF GMCSF plasmid, containing the mGM-CSF transgene under the control of the hCEF promoter, a bovine growth hormone (BGM) polyA sequence, an R6K origin of replication (containing CpG dinucleotides), and a kanamycin resistance cassette (also containing CpG dinucleotides). (b) Schematic linear representation of the pIC098 CMV GMCSF plasmid, containing the mGM-CSF transgene under the control of the CMV promoter, a bovine growth hormone (BGM) polyA sequence, an R6K origin of replication (containing CpG dinucleotides), and a kanamycin resistance cassette (also containing CpG dinucleotides). [Figure 2-1]Figures a-i show schematic diagrams of exemplary plasmids used in the production of exemplary lentiviral vectors of the present invention. (a) A schematic diagram of the lentiviral vector genome plasmid (pDNA1ta+) encoding mGM-CSF and a transactivator under the control of an inducible promoter for lentiviral production of a one-vector system (pSIV-V1-GMCSF). (b) A schematic diagram of the lentiviral vector genome plasmid (pDNA1) encoding mGM-CSF under the control of an inducible promoter for lentiviral production of a two-vector system (pSIV-V2-GMCSF). (c) A schematic diagram of the lentiviral vector genome plasmid (pDNA1*) encoding a transactivator for lentiviral production of a two-vector system (pSIV-V2-transactivator). (d) A schematic diagram of the plasmid (pDNA2a) encoding codon-optimized SIV Gag and Pol for lentiviral production (pGM691). (e) A schematic diagram of the plasmid (pDNA2a) encoding SIV Gag and Pol for lentiviral production (pGM297). (f) Schematic diagram of a plasmid (pDNA2b) encoding SIV Rev for lentivirus production (pGM299). (g) Schematic diagram of a plasmid (pDNA3a) encoding a fusion protein derived from Sendai virus for lentivirus production (pGM301). (h) Schematic diagram of a plasmid (pDNA3b) encoding a hemagglutinin-neuraminidase protein derived from Sendai virus for lentivirus production (pGM303). (i) Schematic diagram of a plasmid (pDNA3) encoding a VSV glycoprotein for lentivirus production (pMD2.G). [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above. [Figure 2-5] Same as above. [Figure 2-6] Same as above. [Figure 2-7] Same as above. [Figure 2-8] Same as above. [Figure 2-9]Same as above. [Figure 3] This graph shows GM-CSF expression by a nonviral expression vector (green bars) at 1, 2, and 6 months after a single treatment compared to GM-CSF expression in the group treated with 1 x 106 TU / mouse lentivirus as a reference (gray bars). Data are presented as median ± IQR (n = 7-8 mice / group). Compared to Glux control by Kruskal-Wallis test with Dunnett's correction for multiple comparisons. *p<0.05. [Figure 4] This graph shows the duration of treatment effect after a single dose of GL67A / mGM-CSF pDNA. GM-CSF knockout mice were treated with the GL67A-mGM-CSF pDNA complex at a dose of 80 μg / mouse. Untreated wild-type mice are included as a reference. Animals were sacrificed 1 to 10 months after transfection, and mGM-CSF expression was quantified in (A) lung homogenates. The effect of mGM-CSF expression on the biomarkers of PAP, (B) BALF turbidity measured by absorbance, (C) SP-D concentration in lung homogenates, (D) surfactant protein D (SP-D) concentration in BALF, and (E) surfactant deposition in alveoli quantified as the percentage of PAS-positive alveoli was analyzed. Data are presented as median ± interquartile range (n = 3-5 per group). Kruskal-Wallis test with Dunnett's correction for multiple comparisons of treated versus untreated control groups. *p<0.05, **p<0.01. [Figure 5]Graphs showing (A) mGM-CSF expression in lung homogenates; (B) surfactant protein D (SP-D) concentration in lung homogenates; (C) BALF turbidity measured by absorbance; and (D) surfactant deposition in alveoli quantified as the percentage of PAS-positive alveoli in GM-CSF KO mice treated with either a single dose (1x) or five doses (5x) of the GL67A-mGM-CSFpDNA complex (80 µg / mouse per dose). Horizontal lines represent the median ± interquartile range (n = 3-5 mice per group). Kruskal-Wallis test with Dunnett's correction for multiple comparisons of treated vs. control groups. *p<0.05, **p<0.01. [Figure 6] Figure 1 shows transient mGM-CSF expression using an inducible promoter. Fully differentiated human air-liquid interface (ALI) cultures were transduced with 1V-GM-CSF at an MOI of 100 and 2V-GM-CSF transgene:transactivator at an MOI of 100:200, and transgene expression was induced with 10-8 M mifepristone for 48 hours. GM-CSF expression was measured in apical lavage fluid for 5 consecutive days. N=3 ALIs / condition. [Figure 7] 1 is a graph showing GM-CSF expression in mouse lungs at days 2 and 22 after administration of pDNA encoding GM-CSF driven by a CMV promoter. GM-CSF expression was measured in lung homogenates by Elisa and normalized for total protein. Data are presented as median ± interquartile range. n=7 mice per group. [Figure 8] (A) SDS-page of purified anti-mouse GM-CSF antibody. The antibody represents both heavy and light chains. The antibody concentration was 820 μg / ml, and the endotoxin level was below the level recommended for animal work (0.67 ng endotoxin per mg antibody). (B) Standard curve of anti-GM-CSF antibody (B2.6) measured by ELISA (OD450 nm vs. B2.6 concentration). The regression equation was y = 0.0045x2 + 0.1782x + 0.1549, and the calculated regression coefficient (R2) was 0.9962. [Figure 9-1]

[0033] Figure 1. Passive immunization of mice with anti-GM-CSF antibody (B2.6). Wild-type mice were treated with different doses of B2.6 antibody: 10, 40, or 80 μg / mouse. Mice were sacrificed b) 1 day (day 1) or c) 7 days (day 7) after a single dose, or d) 1 day after re-dosing (day 1). Antibodies were detected in ELF by ELISA. The black dashed line indicates either the minimum literate threshold (4 μg / ml ELF) required for the development of aPAP disease or the maximum recorded titer reported in aPAP patients. The gray dashed line represents the median antibody titer in two different aPAP cohorts. [Figure 9-2] Same as above. [Figure 10] Passive immunization of mice with anti-GM-CSF antibody (A7.39) was performed at 40 μg / mouse. Antibody levels were measured at 1 day (day 1), 7 days (day 7), or after rechallenge (day post-rechallenge 1) to determine the antibody half-life and to elucidate the antibody dosing schedule required to maintain the median antibody titer from aPAP patients. The black dashed line indicates the minimum threshold (4 μg / ml ELF) required for disease onset. The gray dashed lines represent the median antibody titers in different aPAP cohorts. [Figure 11] This graph shows the results of in vitro neutralization of GM-CSF by the antibody pair (B2.6 and A7.39). Neutralization of GM-CSF (calculated as the percentage of inhibition of FDC-P1 growth using the following formula: [1 - (OD of a single well - mean OD of control cells grown without GM-CSF) x (mean OD of control cells grown with GM-CSF - mean OD of control cells grown without GM-CSF) - 1] x 100) was dose-dependent. Data are presented as median ± interquartile range. n = 6-12 wells per group. [Figure 12] Graph showing luciferase reporter gene expression using different GL67A / mRNA formulations. Total protein concentration and luciferase expression were quantified. All data are expressed as relative light units (RLU) / mg of total protein. Each point represents an individual well. DETAILED DESCRIPTION OF THE INVENTION

[0026] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20th ED., John Wiley and Sons, New York (1994) and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide those skilled in the art with general terminology explanations for many of the terms used in this disclosure. The meaning and scope of the terms should be clear; however, in the event of potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. It should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents, etc., described herein, and as such may vary.

[0027] The present disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0028] The descriptions of embodiments of the present disclosure are not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples of, the present disclosure have been described herein for illustrative purposes, those skilled in the art will recognize that various equivalent modifications are possible within the scope of the present disclosure. For example, while method steps or functions are presented in a given order, in alternative embodiments, the functions may be performed in a different order, or may be performed substantially simultaneously. The teachings of the present disclosure provided herein can be applied to other procedures or methods, as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified, as necessary, to utilize the compositions, functions, and concepts of the above references and applications to provide still further embodiments of the present disclosure. Furthermore, due to considerations of biological functional equivalence, some changes can be made to protein structure without affecting biological or chemical action in terms of nature or quantity. These and other modifications can be made to the present disclosure in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0029] Unless otherwise indicated, any nucleic acid sequence is written left to right in 5' to 3' orientation; an amino acid sequence is written left to right in amino to carboxy orientation, respectively.

[0030] The headings provided herein are not intended to limit the various aspects or embodiments of the disclosure.

[0031] As used herein, the term "capable of" used with a verb encompasses or means the action of the corresponding verb. For example, "capable of interacting" also means interacting, "capable of cleaving" also means cleaving, "capable of binding" also means binding, and "capable of specifically targeting..." also means specifically targeting.

[0032] Other definitions of terms may appear throughout the specification. Before describing exemplary embodiments in more detail, it is to be understood that the present disclosure is not limited to the particular embodiments described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and that the scope of the present disclosure is defined only by the appended claims, and that the terminology is not intended to be limiting.

[0033] Numerical ranges are inclusive of the numbers defining the range. When a range of values ​​is provided, unless the context clearly indicates otherwise, it is understood that each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, is also specifically disclosed. Each smaller range between any stated value or value intermediate in a stated range and any other stated value or value intermediate in that stated range is encompassed within the present disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded therefrom, and each range in which either, neither, or both limits are included in the smaller range is also encompassed within the present disclosure, subject to any specifically excluded limits in the stated range. When a stated range includes one or both of its limits, ranges excluding either or both of those included limits are also encompassed within the present disclosure.

[0034] As used herein, the articles "a" and "an" may refer to one or to more than one (e.g., at least one) of the grammatical object of the article. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. In this application, the use of "or" means "and / or" unless otherwise indicated. Furthermore, the use of the term "including," as well as other forms such as "includes" and "included," is not limiting.

[0035] "About" can generally refer to an acceptable degree of error for a measured number, given the nature or accuracy of the measurement. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically within 5% of a given value or range of values. Preferably, the term "about" is understood herein as plus or minus (±) 5% of the numerical value of the number with which it is used, preferably ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.1%.

[0036] The term "consisting of" refers to the compositions, methods, and their respective components described herein, which exclude any element not recited in that description of the invention.

[0037] As used herein, the term "consisting essentially of" refers to those elements required for a given invention. The term permits the presence of elements that do not materially affect the basic, novel, or functional characteristics of the invention (i.e., inert or non-immunogenic components).

[0038] Embodiments described herein as "comprising" one or more features may also be considered to disclose corresponding embodiments "consisting of" and / or "consisting essentially of" such features.

[0039] Concentrations, amounts, volumes, percentages, and other numerical values ​​may be presented herein in a range format, with the understanding that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values ​​expressly recited as range limits, but also all individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were expressly recited.

[0040] A "vector" or "construct" (sometimes also referred to as a gene delivery or gene transfer "vehicle") refers to a macromolecule or complex of molecules comprising a polynucleotide to be delivered to a host cell either in vitro or in vivo. A vector may be a linear or circular molecule. The vectors of the present invention may be viral or non-viral. All disclosures herein regarding the vectors of the present invention apply equally to viral and non-viral vectors, unless otherwise indicated. All disclosures regarding the viral vectors of the present invention apply equally and without reservation to lentiviral (e.g., SIV) vectors, particularly lentiviral (e.g., SIV) vectors pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxoviruses (also referred to herein as SIV F / HN or SIV-FHN).

[0041] As used herein, the term "viral vector" refers to any viral particle that can be used to deliver genetic material to target cells, including both in vivo and in vitro delivery. The term "viral vector" encompasses both retroviral and lentiviral vectors. All disclosures herein regarding the viral vector of the present invention apply equally and without reservation to the retroviral / lentiviral vector of the present invention, and all disclosures herein regarding the retroviral / lentiviral vector of the present invention apply equally and without reservation to the viral vector of the present invention.

[0042] As used herein, the terms "retroviral vector" and "retroviral F / HN vector" are used interchangeably to refer to a retroviral vector comprising a retroviral RNA sequence and pseudotyped with the hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus, unless otherwise indicated. The terms "lentiviral vector" and "lentiviral F / HN vector" are used interchangeably to refer to a lentiviral vector pseudotyped with the hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus, unless otherwise indicated. All disclosures herein regarding the retroviral vectors of the present invention apply equally and without reservation to the lentiviral vectors of the present invention and to SIV vectors pseudotyped with the hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus (also referred to herein as SIV F / HN or SIV-FHN).

[0043] The term "intron" as used herein refers to the nucleic acid sequence in a gene that is located between exons.Introns are transcribed together with exons, but are removed from the primary gene transcript by RNA splicing to leave behind mature mRNA.Removal of introns typically leads to mRNA stabilization and increases the amount of mRNA in cells.

[0044] As used herein, the term "plasmid" refers to a general type of non-viral vector.Plasmid is an extrachromosomal DNA molecule that can replicate independently of chromosomal DNA and is separate from chromosomal DNA.Preferably, plasmid is circular and can be double-stranded.

[0045] The terms "nucleic acid cassette," "nucleic acid construct," "expression cassette," and "nucleic acid expression cassette" are used interchangeably to refer to a nucleic acid molecule capable of inducing transcription. A nucleic acid cassette contains at least a promoter or a structure functionally equivalent to a promoter and a nucleic acid sequence to be transcribed. Thus, a nucleic acid cassette contains at least a promoter or a structure functionally equivalent to a promoter and a nucleic acid sequence encoding a protein of interest. In the present invention, a nucleic acid cassette contains at least a promoter or a structure functionally equivalent to a promoter, a nucleic acid sequence encoding a signal peptide, and a nucleic acid encoding a therapeutic protein. A nucleic acid cassette may contain additional elements, such as an enhancer and / or a transcription termination signal.

[0046] As used herein, the terms "signal peptide," "signal sequence," "targeting sequence," "leader sequence," and "secretion signal" are used interchangeably to refer to a heterologous peptide sequence found at the N-terminus of a secreted protein that serves to initiate the secretion process. Specifically, signal peptides are found in proteins, particularly single-pass transmembrane proteins, that are targeted to the endoplasmic reticulum and ultimately destined for secretion or retention in the plasma membrane of a cell. The signal peptide is typically removed to produce the mature form of the protein. Signal peptides are usually short peptides, typically about 5 to about 40 amino acids in length, e.g., about 5 to about 35 or about 10 to about 35 amino acids in length, preferably about 10 to about 30 or about 15 to about 30 amino acids in length. Signal peptides may contain a core of hydrophobic amino acids, typically about 4 to about 20, e.g., about 5 to about 20, about 5 to about 16, or about 5 to about 15 amino acids in length. If present, the signal peptide is typically at the N-terminus of the protein.

[0047] As used herein, the terms "transduced" and "modified" are used interchangeably to describe cells that have been modified to express a transgene of interest. Typically, the modification occurs through the transduction of the cells.

[0048] As used herein, the terms "titer" and "yield" are used interchangeably to refer to the amount of viral / retroviral / lentiviral (e.g., SIV) vector produced by the methods of the present invention. Titer is a key benchmark for characterizing production efficiency, with a higher titer generally indicating that more viral / retroviral / lentiviral (e.g., SIV) vector is produced (e.g., using the same amount of reagents). Titer or yield can be related to the number of vector genomes integrated into the genome of target cells (integration titer), which is a measure of "active" viral particles, i.e., the number of particles capable of transducing cells. Transducing units (TU / mL, also referred to as TTU / mL) are a biological readout of the number of host cells transduced under certain tissue culture / virus dilution conditions and are a measure of the number of "active" viral particles. The total number of viral particles (active + inactive) can also be determined by any appropriate means, such as measuring either the amount of Gag present in the test solution or the copy number of viral RNA in the test solution. An assumption is then made that each lentiviral particle contains either 2000 Gag molecules or two viral RNA molecules. After measuring the total particle number and transduction titer / TU, the particle:infectivity ratio is calculated. Amino acids are referred to herein using the amino acid name, three-letter abbreviation, and one-letter abbreviation.

[0049] As used herein, the terms "protein" and "polypeptide" are used interchangeably to refer to a series of amino acid residues connected to each other by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues. The terms "protein" and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. "Protein" and "polypeptide" are often used in reference to relatively large polypeptides, while the term "peptide" is often used to refer to small polypeptides; however, the use of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to gene products and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants, fragments, and analogs of the foregoing.

[0050] As used herein, the terms "polynucleotide," "nucleic acid," and "nucleic acid sequence" refer to any molecule, preferably a polymeric molecule, that incorporates ribonucleic acid, deoxyribonucleic acid, or analog units thereof. A nucleic acid may be single-stranded or double-stranded. A single-stranded nucleic acid may be one nucleic acid strand of denatured double-stranded DNA. Alternatively, it may be a single-stranded nucleic acid that is not derived from any double-stranded DNA. In one embodiment, a nucleic acid may be DNA. In another embodiment, a nucleic acid may be RNA. A suitable nucleic acid molecule is DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including siRNA, shRNA, and antisense oligonucleotides. The terms "transgene" and "gene" are also used interchangeably, and both terms encompass fragments or variants thereof that encode target proteins.

[0051] A transgene of the present invention includes a nucleic acid sequence that has been removed from its naturally occurring environment, a recombinant or cloned DNA isolate, as well as a chemically synthesized analog or a biologically synthesized analog produced by a heterologous system.

[0052] Minor variations in the amino acid sequences of the present invention are intended to be encompassed by the present invention, provided that the variations in the amino acid sequences maintain at least 60%, at least 70%, more preferably at least 80%, at least 85%, at least 90%, at least 95%, and most preferably at least 97% or at least 99% sequence identity with the amino acid sequences of the present invention or their fragments, as defined elsewhere herein. The term "homology" is used herein to mean identity. Therefore, the sequence of a variant or analog sequence of the amino acid sequence of the present invention may differ based on substitution (typically conservative substitution), deletion, or insertion. Proteins containing such variations are referred to herein as variants.

[0053] Proteins of the invention may include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at conserved or non-conserved positions. Variants of the protein molecules disclosed herein may be produced and used in the present invention. Following the lead of computational chemistry in applying multivariate data analysis techniques to structure / property-activity relationships [see, e.g., Wold et al., Multivariate data analysis in chemistry. Chemometrics-Mathematics and Statistics in Chemistry (Ed.: B. Kowalski); D. Reidel Publishing Company, Dordrecht, Holland, 1984 (ISBN 90-277-1846-6)], quantitative activity-property relationships for proteins can be derived using well-known mathematical techniques, such as statistical regression, pattern recognition, and classification [see, e.g., Norman et al., Applied Regression Analysis. Wiley-Interscience; 3rd Edition (April 1998) ISBN: 0471170828; Kandel, Abraham et al., Computer-Assisted Reasoning in Cluster Analysis. Prentice Hall PTR, (May 11, 1995), ISBN: 0133418847; Krzanowski, Wojtek. Principles of Multivariate Analysis: A User's Perspective (Oxford Statistical Science Series, No. 22 (Paper)). Oxford University Press; (December 2000), ISBN: 0198507089; Witten, Ian H. et al., Data Mining: Practical Machine Learning Tools and Techniques with Java Implementations. Morgan Kaufmann; (October 11, 1999), ISBN: 1558605525; Denison David GT(editors), 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 likely contact residues or calculated physicochemical properties), and these properties can be considered individually and in combination.

[0054] Amino acids are referred to herein using the amino acid name, three-letter abbreviation, or single-letter abbreviation. The term "protein," as used herein, includes proteins, polypeptides, and peptides. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "protein." In some instances, the term "amino acid sequence" is synonymous with the term "peptide." The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, conventional single-letter and three-letter codes for amino acid residues may be used. The three-letter code for amino acids is as defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide can be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0055] Amino acid residues at non-conserved positions may be substituted with either conservative or non-conserved residues. Conservative amino acid substitutions are particularly contemplated.

[0056] "Conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, or histidine), acidic side chains (e.g., aspartic acid or glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, or histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the amino acid substitution is considered conservative. The inclusion of conservatively modified variants in the proteins of the invention does not exclude other forms of variants, such as polymorphic variants, interspecies homologs, and alleles.

[0057] "Non-conservative amino acid substitutions" include (i) substitutions of a positively charged side chain (e.g., Arg, His, or Lys) with or by a negatively charged residue (e.g., Glu or Asp), (ii) substitutions of a hydrophilic residue (e.g., Ser or Thr) with or by a hydrophobic residue (e.g., Ala, Leu, Ile, Phe, or Val), (iii) substitutions of a cysteine ​​or proline with or by any other residue, or (iv) substitutions of a residue with a bulky hydrophobic or aromatic side chain (e.g., Val, His, Ile, or Trp) with or by one with a smaller side chain (e.g., Ala or Ser) or no side chain (e.g., Gly).

[0058] "Insertions" or "deletions" are typically within the range of about 1, 2, or 3 amino acids. Tolerable variations can be determined experimentally by using recombinant DNA techniques to systematically introduce amino acid insertions or deletions into a protein and assaying the resulting recombinant variants for activity. This does not require more experimentation than is routine for one of ordinary skill in the art.

[0059] A "fragment" of a polypeptide comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or more of the original polypeptide.

[0060] The polynucleotide of the present invention can be prepared by any means known in the art.For example, large amounts of polynucleotide can be produced by replication in suitable host cells.The natural or synthetic DNA fragment encoding the desired fragment will be incorporated into a recombinant nucleic acid construct, typically a DNA construct, that can be integrated into and replicated in prokaryotic or eukaryotic cells.Usually, the DNA construct is suitable for autonomous replication in unicellular hosts, such as yeast or bacteria, but it is also intended to be introduced into cultured insects, mammals, plants, or other eukaryotic cell systems and integrated into their genomes.

[0061] Polynucleotides of the invention can also be produced by chemical synthesis, for example, by the phosphoramidite or triester method, which can be performed using commercially available automated oligonucleotide synthesizers. Double-stranded fragments can be obtained from the single-stranded product of chemical synthesis by either synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

[0062] The term "isolated" in the context of the present invention, when applied to a nucleic acid sequence, indicates that the polynucleotide sequence has been removed from its natural genetic environment and therefore is free of other unrelated or undesired coding sequences (although naturally occurring 5' and 3' untranslated regions, e.g., promoters and terminators, may be included), and is in a form suitable for use in a genetically engineered protein production system. Such isolated molecules are separated from their natural environment.

[0063] Given the degeneracy of the genetic code, considerable sequence variation is possible among the polynucleotides of the present invention. Degenerate codons that encompass all possible codons for a given amino acid are listed below:

[0064] [Table 1]

[0065] Those skilled in the art will recognize that there is flexibility in determining degenerate codons that represent all possible codons that encode each amino acid. For example, some polynucleotides encompassed by a degenerate sequence may encode variant amino acid sequences, and those skilled in the art can readily identify such variant sequences by reference to the amino acid sequences of the present invention.

[0066] A "variant" nucleic acid sequence has substantial homology or substantial similarity to a reference nucleic acid sequence (or a fragment thereof). A nucleic acid sequence or a fragment thereof is "substantially homologous" (or "substantially identical") to a reference sequence if, when optimally aligned with another nucleic acid (or its complementary strand) (with appropriate nucleotide insertions or deletions), there is at least about 70%, 75%, 80%, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more of the nucleotide bases have nucleotide sequence identity. Methods for determining the homology of nucleic acid sequences are known in the art.

[0067] Alternatively, a "variant" nucleic acid sequence is substantially homologous to (or substantially identical to) a reference sequence (or a fragment thereof) if the "variant" and the reference sequence are capable of hybridizing under stringent (e.g., highly stringent) hybridization conditions. Hybridization of nucleic acid sequences will be affected by conditions such as salt concentration (e.g., NaCl), temperature, or organic solvents, in addition to base composition, length of complementary strands, and number of nucleotide base mismatches between hybridizing nucleic acids, as will be readily recognized by those skilled in the art. Stringent temperature conditions are preferably used, generally including temperatures above 30°C, typically above 37°C, and preferably above 45°C. Stringent salt conditions will usually be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. pH is typically between 7.0 and 8.3. The combination of parameters is much more important than any single parameter.

[0068] The method for determining nucleic acid sequence identity percentage is known in the art.For example, when evaluating nucleic acid sequence identity, the sequence with a predetermined number of consecutive nucleotides can be aligned with the nucleic acid sequence (with the same number of consecutive nucleotides) from the corresponding part of the nucleic acid sequence of the present invention.The tool known in the art for determining nucleic acid sequence identity percentage includes nucleotide BLAST (described below).

[0069] Those skilled in the art understand that different species exhibit "preferential codon usage." As used herein, the term "preferential codon usage" refers to the codons most frequently used in cells of a particular species, and thus one or several representative codons that may encode each amino acid are preferred. For example, the amino acid threonine (Thr) is encoded by ACA, ACC, ACG, or ACT, but in mammalian host cells, ACC is the most commonly used codon; in other species, different codons may be preferred. Preferred codons for a particular host cell species can be introduced into the polynucleotides of the present invention by various methods known in the art. Introducing preferred codon sequences into recombinant DNA can enhance protein production, for example, by making protein translation more efficient within a particular cell type or species. Thus, according to the present invention, any nucleic acid sequence, in addition to the gag-pol gene, can be codon-optimized for expression in a host or target cell. Specifically, the vector genome (or corresponding plasmid), the REV gene (or corresponding plasmid), the fusion protein (F) gene (or corresponding plasmid), and / or the hemagglutinin-neuraminidase (HN) gene (or corresponding plasmid), or any combination thereof, may be codon-optimized.

[0070] A "fragment" of a polynucleotide of interest comprises a series of contiguous nucleotides derived from the sequence of said full-length polynucleotide. By way of example, a "fragment" of a polynucleotide of interest may comprise (or consist of) at least 30 contiguous nucleotides derived from the sequence of said polynucleotide (e.g., at least 35, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 contiguous nucleic acid residues of said polynucleotide). A fragment may comprise at least one antigenic determinant and / or encode at least one antigenic epitope of the corresponding polypeptide of interest. Typically, a fragment as defined herein retains the same function as the full-length polynucleotide.

[0071] The terms "reduce," "reduced," "reduction," or "inhibit" are all used herein to mean a statistically significant reduction. The terms "reduce," "reduction," "reducing," or "inhibiting" typically refer to a reduction of at least 10% compared to a reference level (e.g., in the absence of a given treatment), and can include, for example, a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, "reduction" or "inhibition" encompasses complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition (i.e., abolition) compared to a reference level.

[0072] The terms "increased," "increase," "enhance," or "activate" are all used herein to mean an increase by a statistically significant amount. The terms "increased," "increase," "enhance," or "activate" can mean an increase of at least 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 a reference level, or an increase of at least about 1.5-fold, or at least about 2-fold, or at least about 2.5-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase from 1.5-fold to 10-fold or more, compared to a reference level. In the context of yield or titer, an "increase" is an observable or statistically significant increase in such level.

[0073] The terms "individual," "subject," and "patient" are used interchangeably herein to refer to a mammalian subject for whom diagnosis, prognosis, disease monitoring, treatment, therapy, and / or therapy optimization is desired. The mammal may be (without limitation) a human, non-human primate, mouse, rat, dog, cat, horse, or cow. In preferred embodiments, the individual, subject, or patient is human. An "individual" may be an adult, juvenile, or child. An "individual" may be male or female.

[0074] A "subject in need" of treatment for a particular condition can be an individual who has the condition, has been diagnosed with the condition, or is at risk of developing the condition.

[0075] The subject may be a subject who has been previously diagnosed or identified as suffering from or having a condition requiring treatment or one or more co-morbidities or symptoms associated with such a condition, and may optionally have already been treated for a condition as defined herein or one or more co-morbidities or symptoms associated with said condition. Alternatively, the subject may also be a subject who has not previously been diagnosed with a condition as defined herein or one or more symptoms or co-morbidities associated with said condition. For example, the subject may be a subject who exhibits one or more risk factors for a condition or one or more symptoms or co-morbidities associated with said condition, or may be a subject who does not exhibit risk factors.

[0076] As used herein, the term "healthy individual" refers to an individual or group of individuals in a healthy state, for example, an individual who does not show any symptoms of a disease, has not been diagnosed with a disease, and / or is not likely to develop a disease, such as aPAP or any other disease described herein.Preferably, the healthy individual does not take a drug that affects aPAP or has not been diagnosed with any other disease.One or more healthy individuals may have similar gender, age, and / or body mass index (BMI) compared with the test individual.By applying standard statistical methods used in medicine, it is possible to determine normal expression levels in healthy individuals and significant deviations from such normal levels.

[0077] As used herein, the terms "control" and "reference population" are used interchangeably.

[0078] The term "pharmaceutically acceptable," as used herein, means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias.

[0079] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present patent application, and nothing herein should be construed as an admission that such publications constitute prior art to the appended claims.

[0080] It is intended that disclosure relating to various methods of the invention applies equally to other methods, therapeutic uses or methods, and vice versa.

[0081] Treatment of pulmonary alveolar proteinosis (PAP) The present invention relates to the treatment of pulmonary alveolar proteinosis (PAP). PAP is a disease of alveolar filling characterized by dense phospholipid proteinaceous deposits in the alveoli, cough, and shortness of breath. This disease is often associated with impaired processing of pulmonary surfactant by alveolar macrophages, a process dependent on granulocyte-macrophage colony-stimulating factor (GM-CSF). PAP has three distinct pathogenic mechanisms: hereditary, autoimmune, and secondary. Approximately 90-95% of PAP cases are autoimmune, in which high levels of autoantibodies against GM-CSF neutralize its biological activity, thereby causing impaired surfactant clearance. The present invention relates to the treatment of PAP, particularly autoimmune PAP (aPAP). In aPAP, the minimum threshold of autoantibodies to GM-CSG that can cause disease onset has been described in the art as 4 μg / mL in epithelial lining fluid (ELF) (see Sakagami et al., Am J Respir Crit Care Med. 2010 Jul 1;182(1):49-61, which is incorporated herein by reference in its entirety).

[0082] Treatment according to the present invention provides clinical benefits to patients. Treatment according to the present invention can be defined as providing any one or more of the following defined treatment results. These definitions apply to the therapeutic and prophylactic treatments described herein. These treatment biomarkers (e.g., BALF turbidity; SF-D concentration in the lungs and / or BALF; surfactant deposition; lung pathology, such as (i) lung opacity, (ii) pulmonary edema, and / or (iii) lung consolidation; and / or lung function, such as (i) VC, (ii) FVC, and / or (iii) FEV (e.g., FEV1)) can be considered biomarkers of PAP, particularly aPAP.

[0083] Treatment of PAP, particularly aPAP, according to the present invention can reduce the turbidity of BALF obtained from a patient and / or the duration and / or volume of lavage fluid required to clear the BALF. Specifically, treatment can reduce BALF turbidity, lavage duration, and / or lavage fluid volume by at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, or more. Preferably, there is a reduction in BALF turbidity of at least 30%, more preferably at least 40%. The reduction in BALF turbidity can be compared with a suitable control, such as the turbidity of BALF obtained from a healthy individual or the turbidity of BALF obtained from a patient before treatment according to the present invention. Any suitable method can be used to assess or quantify BALF turbidity. Standard techniques are known in the art and can be readily used by those skilled in the art without undue burden. As a non-limiting example, BALF turbidity can be analyzed by absorbance at 600 nm or visually determined by the clinician performing the lavage.

[0084] Alternatively, or in addition, treatment of PAP, particularly aPAP, according to the present invention can reduce the concentration of surfactant protein D (SF-D) in the patient's lungs and / or BALF. Specifically, treatment can reduce the concentration of surfactant protein D (SF-D) in the lungs and / or BALF by at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, or more. Preferably, there is a reduction in BALF turbidity of at least 30%, more preferably at least 40%. The reduction in SF-D concentration in the patient's lungs and / or BALF can be compared with a suitable control, such as the SF-D concentration in the lungs and / or BALF of a healthy individual, or the SF-D concentration in the patient's lungs and / or BALF before treatment according to the present invention. Any suitable method can be used to assess or quantify SF-D concentration in the lungs and / or BALF. Standard techniques are known in the art and can be readily used by those skilled in the art without undue burden. As a non-limiting example, SF-D concentrations in the lungs and / or BALF can be analyzed by ELISA.

[0085] Alternatively or additionally, the treatment of PAP, particularly aPAP, according to the present invention can reduce surfactant deposition in the lungs of patients, particularly in the alveoli. Specifically, the treatment can reduce surfactant deposition in the lungs, particularly in the alveoli, by at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60% or more. Preferably, there is a reduction of at least 30%, more preferably at least 40% in surfactant deposition in the lungs, particularly in the alveoli. The reduction of surfactant deposition in the lungs of patients can be compared with a suitable control, for example, surfactant deposition in the lungs of healthy individuals, or surfactant deposition in the lungs of patients before the treatment according to the present invention. Standard techniques are known in the art and can be easily used by those skilled in the art without undue burden. As a non-limiting example, surfactant deposition in the lung may be analyzed by periodic acid-Schiff (PAS) staining, which detects polysaccharides and mucous substances such as surfactant.

[0086] Alternatively or additionally, treatment of PAP, particularly aPAP, according to the present invention can reduce lung pathology, for example, reducing lung opacity, pulmonary edema, and / or lung consolidation in a patient. Specifically, treatment can reduce lung opacity by at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, or more. Preferably, there is a reduction in lung opacity of at least 30%, more preferably at least 40%. Specifically, treatment can reduce pulmonary edema by at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, or more. Preferably, there is a reduction in pulmonary edema of at least 30%, more preferably at least 40%. Specifically, treatment can reduce lung consolidation by at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, or more. Preferably, there is a reduction in lung consolidation of at least 30%, more preferably at least 40%. The reduction in lung pathology in a patient, for example, the reduction in lung opacity, pulmonary edema, and / or lung consolidation, may be compared with a suitable control, for example, lung pathology such as lung opacity, pulmonary edema, and / or lung consolidation in the lungs of a healthy individual, or lung pathology such as lung opacity, pulmonary edema, and / or lung consolidation in the lungs of a patient before treatment according to the present invention. Standard techniques are known in the art and can be easily used by those skilled in the art without undue burden. As a non-limiting example, lung pathology in the lung, for example, lung opacity, pulmonary edema, and / or lung consolidation, can be detected by imaging, for example, highly sensitive imaging techniques including computed tomography (CT) and / or magnetic resonance imaging (MRI).

[0087] Alternatively, or in addition, the treatment of PAP, particularly aPAP, according to the present invention can increase a patient's pulmonary function. Numerous measures of pulmonary function exist, including vital capacity (VC), forced vital capacity (FVC), forced expiratory volume (FEV); arterial oxygen tension (Pa,O2); and alveolar-arterial oxygen gradient (PA-a,O2). Other measures of pulmonary function include peak metabolic equivalents (peak METS) and / or 6-minute walking distance (6MWD). One or more of these parameters may be measured at appropriate intervals. As a non-limiting example, FEV1 over 1 second (FEV1) is particularly preferred. Even relatively small numerical improvements can have a significant impact on a patient's quality of life. Thus, treatment can increase VC, FVC, and / or FEV (e.g., FEV1) by at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 15%, at least 20%, at least 25%, or more. Alternatively, or in addition, Pa,O2 and / or PA-a,O2 can increase by at least about 5 mmHg, at least about 6 mmHg, at least about 7 mmHg, at least about 8 mmHg, at least about 9 mmHg, at least about 10 mmHg, at least about 11 mmHg, at least about 12 mmHg, at least about 13 mmHg, at least about 14 mmHg, at least about 15 mmHg, or more. Alternatively, or in addition, peak METS can increase by at least about 2 METS, at least about 3 METs, or at least about 4 METS. Alternatively, or in addition, 6MWD may increase by at least about 100 m, at least about 150 m, at least about 200 m, at least about 250 m, at least about 300 m, or more. Preferably, there is an increase in VC, FVC, and / or FEV (e.g., FEV1) of at least 5%, more preferably at least 10%. Alternatively, or in addition, Pa,O2 and / or PA-a,O2 may preferably increase by at least about 10 mmHg or at least about 12 mmHg.Alternatively, or in addition, peak METS may preferably increase by at least about 2 METs. Alternatively, or in addition, 6MWD may preferably increase by at least about 200 m. The increases in VC, FVC, FEV (e.g., FEV1), Pa,O2, PA-a,O2, peak METs, and / or 6MWD may be compared with corresponding parameters measured in a suitable control, e.g., a healthy individual or measured in a patient prior to treatment according to the present invention. Standard techniques are known in the art and can be readily used by those skilled in the art without undue burden. As a non-limiting example, VC, FVC, and / or FEV (e.g., FEV1) can be measured by spirometry.

[0088] Suitable controls can be used as described herein. As a non-limiting example, one or more treatment results in an individual treated according to the present invention can be compared with a suitable control, such as the same parameter in a healthy individual, or a parameter in an individual (typically the same individual) with PAP, particularly aPAP, before treatment. Any one or more of these treatment results can be measured at one or more time points after treatment and compared with one or more corresponding parameters in the patient before treatment. As a non-limiting example, any one or more of these treatment results can be compared with one or more corresponding parameters in the patient before treatment at 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks, 28 weeks, or more, preferably 24 weeks, after treatment.

[0089] Any combination of the above-mentioned (a) BALF turbidity, or the lavage duration and / or lavage volume required to produce clear BALF; (b) SF-D concentration in the lung; (c) SF-D concentration in the BALF; (d) surfactant deposition; (e) pulmonary pathology, such as (i) pulmonary opacity, (ii) pulmonary edema, and / or (iii) pulmonary consolidation; and / or (f) pulmonary function, such as (i) VC, (ii) FVC, (iii) FEV (e.g., FEV1); (iv) Pa,O2, (v) PA-a,O2, (vi) peak METs, and / or (vii) 6MWD, can be evaluated, quantified, or determined to assess treatment according to the present invention. As non-limiting examples, the following combinations can be used: BALF turbidity or the lavage period and / or lavage volume required to obtain clear BALF; and SF-D concentration in the lung (a+b); BALF turbidity or the lavage period and / or lavage volume required to obtain clear BALF; and SF-D concentration in the BALF (a+c); BALF turbidity or the lavage period and / or lavage volume required to obtain clear BALF; and surfactant deposition (a+d); BALF turbidity or the lavage period and / or lavage volume required to obtain clear BALF; and lung pathology (a+e); BALF turbidity or the lavage period and / or lavage volume required to obtain clear BALF. clear fluid volume; and pulmonary function (a+f); SF-D concentration in the lung; and SF-D concentration in BALF (b+c); SF-D concentration in the lung; and surfactant deposition (b+d); SF-D concentration in the lung; and pulmonary pathology (b+e); SF-D concentration in the lung; and pulmonary function (b+f); SF-D concentration in BALF; and surfactant deposition (c+d); SF-D concentration in BALF; and pulmonary pathology (c+e); SF-D concentration in BALF; and pulmonary function (c+f); surfactant deposition; and pulmonary pathology (d+e); surfactant deposition; and pulmonary function (d+f); pulmonary pathology; and pulmonary function (e+f); BALF turbidity or lavage duration and / or lavage fluid volume required to obtain clear BALF;SF-D concentration in the lungs and SF-D concentration in BALF (a+b+c); BALF turbidity or the lavage period and / or lavage volume required to obtain clear BALF; SF-D concentration in the lungs; and surfactant deposition (a+b+d); BALF turbidity or the lavage period and / or lavage volume required to obtain clear BALF; SF-D concentration in the lungs; and lung pathology (a+b+e); BALF turbidity or the lavage period and / or lavage volume required to obtain clear BALF; SF-D concentration in the lungs and pulmonary function (a+b+f); BALF turbidity or the lavage duration and / or lavage volume required to obtain clear BALF; SF-D concentration in BALF; and surfactant deposition (a+c+d); BALF turbidity or the lavage duration and / or lavage volume required to obtain clear BALF; SF-D concentration in BALF; and pulmonary pathology (a+c+e); BALF turbidity or the lavage duration and / or lavage volume required to obtain clear BALF; SF-D concentration in BALF; and pulmonary function (a +c+f); BALF turbidity or the lavage duration and / or lavage volume required to obtain clear BALF; surfactant deposition; and lung pathology (a+d+e); BALF turbidity or the lavage duration and / or lavage volume required to obtain clear BALF; surfactant deposition; and lung function (a+d+f); BALF turbidity or the lavage duration and / or lavage volume required to obtain clear BALF; lung pathology; and lung function (a+e+f); SF-D concentration in the lung; SF-D concentration in BALF; and Surfactant deposition (b+c+d); SF-D concentration in the lung; SF-D concentration in BALF; and lung pathology (b+c+e); SF-D concentration in the lung; SF-D concentration in BALF; and lung function (b+c+f); SF-D concentration in the lung; surfactant deposition; and lung pathology (b+d+e); SF-D concentration in the lung; surfactant deposition; and lung function (b+d+f); SF-D concentration in BALF; surfactant deposition; and lung pathology (c+d+e); SF-D concentration in the lung; lung pathology; and lung function (b+e+f);SF-D concentration in BALF; surfactant deposition; and lung function (c+d+f); SF-D concentration in BALF; lung pathology; and lung function (c+e+f); surfactant deposition; lung pathology; and lung function (d+e+f); BALF turbidity or lavage duration and / or lavage volume required to obtain clear BALF; SF-D concentration in lung; SF-D concentration in BALF; and surfactant deposition (a+b+c+d); BALF turbidity or lavage duration and / or lavage volume required to obtain clear BALF; SF-D concentration in the lung; SF-D concentration in BALF; and lung pathology (a+b+c+e); BALF turbidity or the lavage period and / or lavage volume required to obtain clear BALF; SF-D concentration in the lung; SF-D concentration in BALF; and lung function (a+b+c+f); BALF turbidity or the lavage period and / or lavage volume required to obtain clear BALF; SF-D concentration in the lung; surfactant deposition; and lung pathology (a+b+d+e); BALF turbidity or the lavage period and / or lavage volume required to obtain clear BALF BALF turbidity or the duration and / or volume of lavage fluid required to obtain clear BALF; SF-D concentration in the lungs; surfactant deposition; and pulmonary function (a+b+d+f); BALF turbidity or the duration and / or volume of lavage fluid required to obtain clear BALF; SF-D concentration in the lungs; pulmonary pathology; and pulmonary function (a+b+e+f); BALF turbidity or the duration and / or volume of lavage fluid required to obtain clear BALF; SF-D concentration in the BALF; surfactant deposition; and pulmonary pathology (a+c+d+e); BALF turbidity or the duration and / or volume of lavage fluid required to obtain clear BALF BALF turbidity or the lavage duration and / or lavage volume required to obtain clear BALF; SF-D concentration in BALF; surfactant deposition; and lung function (a+c+d+f); BALF turbidity or the lavage duration and / or lavage volume required to obtain clear BALF; SF-D concentration in BALF; lung pathology; and lung function (a+c+e+f); BALF turbidity or the lavage duration and / or lavage volume required to obtain clear BALF; surfactant deposition; lung pathology; and lung function (a+d+e+f); SF-D concentration in the lung;SF-D concentration in BALF; surfactant deposition; and lung pathology (b+c+d+e); SF-D concentration in lung; SF-D concentration in BALF; surfactant deposition; and lung function (b+c+d+f); SF-D concentration in lung; SF-D concentration in BALF; lung pathology; and lung function (b+c+e+f); SF-D concentration in lung; surfactant deposition; lung pathology; and lung function (b+d+e+f); SF-D concentration in BALF; surfactant deposition; lung pathology; and pulmonary function (c+d+e+f); BALF turbidity or the lavage duration and / or lavage volume required to obtain clear BALF; SF-D concentration in the lung; SF-D concentration in the BALF; surfactant deposition; and pulmonary pathology (a+b+c+d+e); BALF turbidity or the lavage duration and / or lavage volume required to obtain clear BALF; SF-D concentration in the lung; SF-D concentration in the BALF; surfactant deposition; and pulmonary function (a+b+c+d+f ); BALF turbidity or the lavage duration and / or lavage volume required to obtain clear BALF; SF-D concentration in the lungs; SF-D concentration in BALF; lung pathology; and lung function (a+b+c+e+f); BALF turbidity or the lavage duration and / or lavage volume required to obtain clear BALF; SF-D concentration in the lungs; surfactant deposition; lung pathology; and lung function (a+b+d+e+f); BALF turbidity or the lavage duration and / or lavage volume required to obtain clear BALF; SF-D concentration in BALF; surfactant deposition; lung pathology; and lung function (a+c+d+e+f); SF-D concentration in the lungs; SF-D concentration in BALF; surfactant deposition; lung pathology; and lung function (b+c+d+e+f); and BALF turbidity or the lavage duration and / or lavage volume required to obtain clear BALF; SF-D concentration in the lungs; SF-D concentration in BALF; surfactant deposition; lung pathology;and pulmonary function (a+b+c+d+e+f) can be used. In any of the above combinations, when pulmonary pathology (e) is evaluated, quantified, or determined, the pathology evaluated, quantified, or determined can be selected from (i) lung opacity, (ii) lung edema, (iii) lung consolidation, (iv) lung opacity and pulmonary edema, (v) lung opacity and lung consolidation, (vi) pulmonary edema and lung consolidation; or (vii) lung opacity, pulmonary edema, and lung consolidation. Alternatively, or in addition, in any of the above combinations, when pulmonary function (f) is assessed, quantified, or determined, the function assessed, quantified, or determined can be selected from (i) VC, (ii) FVC, (iii) FEV (e.g., FEV1), (iv) Pa,O2, (v) PA-a,O2, (vi) peak METs; (vii) 6MWD; or any combination thereof, with PA-a,O2 or a combination including PA-a,O2 being preferred.

[0090] The treatment of PAP, particularly aPAP, according to the present invention is typically not associated with one or more histopathological changes in patients. Non-limiting examples of such histopathological changes include (a) one or more histopathological changes in the lungs; (b) one or more histopathological changes in the liver; (c) one or more histopathological changes in the kidneys; and / or (d) one or more histopathological changes in the spleen. The treatment of PAP, particularly aPAP, according to the present invention is not associated with any combination of (a), (b), (c), and / or (d).

[0091] Histopathological changes in the lungs that are typically not associated with treatment according to the present invention can include one or more of the following: distortion of lung architecture, pulmonary inflammatory cell infiltration exceeding the PAP phenotype, increased alveolar wall thickening, pulmonary alveolar microlithiasis (PAM) alveoli, PAM bronchi, presence of neutrophils in the bronchi, consolidation, presence of giant cells, eosinophilic material, and / or edema. These histopathological changes can be assessed or determined by any suitable means, including direct and indirect assessment and / or quantification as described herein, e.g., by imaging (e.g., CT scan), pulmonary function testing, or histological analysis.

[0092] Histopathological changes in the liver that are typically not associated with treatment according to the present invention can include one or more of inflammatory cell infiltration, portal tract inflammation, sinusoidal dilated congestion, and / or vascular dilated congestion that exceed the PAP phenotype (e.g., as assessed or quantified in patients prior to treatment). These histopathological changes can be assessed or determined by any suitable means, including direct and indirect assessment and / or quantification as described herein, for example, imaging (e.g., by CT scan), liver function tests, or histological analysis.

[0093] Histopathological changes in the kidney that are typically not associated with treatment according to the present invention can include one or more of inflammatory cell infiltration, vascular dilation, fibrosis, eosinophilic material, and / or cysts above the PAP phenotype (e.g., as assessed or quantified in the patient prior to treatment). These histopathological changes can be assessed or determined by any suitable means, including direct and indirect assessment and / or quantification as described herein, e.g., by imaging (e.g., by CT scan), renal function testing, or histological analysis.

[0094] Histopathological changes in the spleen that are typically not associated with treatment according to the present invention can include one or more of the presence of megakaryocyte clusters and / or macrophages. These histopathological changes can be assessed or determined by any suitable means, including direct and indirect assessment and / or quantification as described herein, for example, imaging (e.g., by CT scan), spleen function tests, or histological analysis.

[0095] GM-CSF expression The present invention provides a gene therapy vector capable of expressing GM-CSF in target cells, as described herein. An exemplary GM-CSF is human GM-CSF, which has UniProt accession number P04141 (version 1, registered November 1, 1986, accessed September 25, 2022) or SEQ ID NO: 1. Therapeutic GM-CSF protein is encoded by the gene CSF2. An example of a human CSF2 transgene is provided in GenBank accession number M11220.1 (version 1, registered November 8, 1994, accessed September 25, 2022), which is SEQ ID NO: 2. Another exemplary GM-CSF is mouse GM-CSF, which has UniProt accession number P01587 (version 1, registered April 1, 1988, accessed September 25, 2022) or SEQ ID NO: 3. An example of a mouse CSF2 transgene is provided in GenBank Accession No. AY950559.1 (Version 1, deposited December 19, 2026, accessed September 29, 2022), which is SEQ ID NO: 4, and another example is SEQ ID NO: 5. Preferably, the GM-CSF is human GM-CSF (hGM-CSF). Preferably, the CSF2 transgene is human CSF2. Also included are variants thereof (described herein), particularly variants having at least 90% (e.g., at least 90, 92, 94, 95, 96, 97, 98, 99, or 100%) identity to SEQ ID NO: 1, 2, 3, 4, or 5, preferably 1 or 2. Any reference herein to a GM-CSF protein may refer to the GM-CSF of SEQ ID NO: 1 or 3, preferably 1, or a functional fragment and / or variant thereof. Any reference herein to a GM-CSF transgene may refer to the GM-CSF transgene of SEQ ID NO: 2, 4 or 5, preferably 2, or a functional fragment and / or variant thereof.

[0096] The therapeutic window (also referred to interchangeably herein as the toxicity / efficacy window) is the concentration range of a drug that achieves a therapeutic effect. Below this range, there is little or no therapeutic benefit, and above this range, unacceptable levels of toxicity occur. The therapeutic window for GM-CSF is narrow. This is because in GM-CSF knockout mice, the therapeutic window is 1×10 5 TU~1×10 6 This is supported by the fact that it is calculated to be in the range of less than TU.

[0097] A widespread teaching in gene therapy is that multiple gene therapy agents must be delivered to achieve a therapeutic effect, prompting research to achieve this goal, including increasing vector yields, increasing transgene expression from the vector, and introducing gain-of-function mutations to increase the potency of therapeutic proteins.

[0098] Contrary to this standard teaching in the art, for the treatment of PAP, particularly aPAP, GM-CSF needs to be expressed in patients within this narrow therapeutic window. Therefore, conventional teachings and gene therapy vectors are not suitable for this indication. Preferably, the level of free GM-CSF needs to be within a narrow therapeutic window. By free GM-CSF, we mean GM-CSF that is not neutralized by autoimmune antibodies against GM-CSF. Methods for determining the neutralization of GM-CSF are routine for those skilled in the art and are exemplified herein, such as the neutralization assay described in Example 8. In aPAP, autoimmune antibodies against GM-CSF may neutralize a portion of the GM-CSF administered to the patient, and therefore not all of the administered GM-CSF is available to perform physiological functions. The level of autoimmune antibodies against GM-CSF may vary between patients. Typically, the present invention seeks to provide sufficient GM-CSF so that the concentration of free GM-CSF in a patient falls within a narrow therapeutic window, resulting in therapeutic benefit without the histopathological changes associated with the administration of high and / or sustained doses of GM-CSF. Accordingly, any reference herein to the therapeutic window of GM-CSF applies equally and without reservation to the therapeutic window of free GM-CSF.

[0099] On the other hand, the present inventors have discovered for the first time that transient and / or low-level expression of GM-CSF (particularly, free GM-CSF) can provide therapeutic benefits without the problems typically associated with higher-level and / or longer-term GM-CSF expression. In this application, the present inventors provide for the first time a gene therapy agent that is capable of inducing GM-CSF expression (particularly, free GM-CSF) within a narrow therapeutic window. Specifically, the gene therapy agent of the present invention allows for careful control of the duration of GM-CSF expression in a patient's cells and / or the concentration of GM-CSF (particularly, free GM-CSF) expressed in the patient's cells, enabling transient and / or low-level expression, resulting in GM-CSF (particularly, free GM-CSF) being expressed within a narrow toxicity / efficacy window.

[0100] Thus, the gene therapy agents of the present invention typically allow transient expression of GM-CSF in a patient (i.e., in the patient's cells into which the agent is introduced). Transient expression of GM-CSF can be defined as expression for 6 months or less, e.g., 5 months or less, 4 months or less, 3 months or less, 2 months or less, 1 month or less, less than 3 weeks, less than 2 weeks, or less. In some preferred embodiments, transient expression of GM-CSF is for about 1 to 6 months, e.g., about 1 to 4 months, 1 to 3 months, 1 to 2 months, 1 week to 4 months, 1 week to 3 months, 1 week to 2 months, or 1 week to 1 month. In some preferred embodiments, transient expression of GM-CSF is for 3 months or less.

[0101] References to the expression of GM-CSF herein apply equally and without reservation to both the expression of the GM-CSF transgene and the expression of the encoded GM-CSF protein, unless expressly indicated otherwise. The expression level of the GM-CSF transgene and / or the encoded GM-CSF protein of the present invention can be measured in lung tissue, epithelial lining fluid, and / or serum / plasma, as appropriate. The therapeutic expression level can therefore refer to the concentration in the lung, epithelial lining fluid, and / or serum / plasma. As described herein, in healthy individuals, the concentration of GM-CSF is typically low, or even below the lower limit of detection using standard assays (e.g., ELISA or other standard protein quantification assays). Therefore, the period of transient GM-CSF (particularly free GM-CSF) expression according to the present invention can be defined as the period during which GM-CSF protein can be detected or during which one or more of the treatment results defined herein are observed.

[0102] As described herein, the viral vectors of the present invention, particularly the retroviral / lentiviral (e.g., SIV) vectors of the present invention, can be integrated into the genome of target cells in a patient. Once integrated, these viral vectors, particularly these retroviral / lentiviral (e.g., SIV) vectors, are maintained in the genome of the target cells for the life of the cells. Thus, although these viral vectors, particularly these retroviral / lentiviral (e.g., SIV) vectors, can be used to induce transient expression of GM-CSF, the vector typically exists (integrated into the genome of) the target cells for a period longer than the period of expression. As a non-limiting example, the viral vectors, particularly the retroviral / lentiviral (e.g., SIV) vectors, can exist in target cells for at least 180 days, at least 250 days, at least 360 days, at least 450 days, at least 720 days, or longer. Using the inducible promoters described herein, such viral vectors, particularly retroviral / lentiviral (e.g., SIV) vectors, can be used to transiently express GM-CSF as described herein for a period of 6 months or less, e.g., 5 months or less, 4 months or less, 3 months or less, 2 months or less, 1 month or less, 3 weeks or less, 2 weeks or less, or less, preferably 3 months or less.

[0103] As described herein, expression of GM-CSF can be induced a single time using the inducible promoter of the present invention (whether for viral or non-viral gene therapy agents). When the gene therapy agent is to be maintained in the target cell for an extended period of time, for example, typically in the case of viral vectors, particularly the retroviral / lentiviral (e.g., SIV) vectors described herein, expression of GM-CSF can be induced multiple times, for example, two, three, four, five, six, seven, eight, nine, or ten or more times, using the inducible promoter.

[0104] Therefore, the gene therapy agent is administered once, retained in target cells, and then used to express GM-CSF for a short period of time. This can maintain the concentration of GM-CSF (especially free GM-CSF) within a narrow therapeutic window, achieving a therapeutic effect on patients, while reducing and / or eliminating the histopathological changes in patients that are usually associated with long-term and / or high-level GM-CSF expression. Without being bound by theory, the viral gene therapy agent of the present invention, for example, a viral vector, particularly a retrovirus / lentivirus (e.g., SIV) vector, is typically integrated into the genome of target cells, thereby being retained in target cells for a long period of time, and can therefore be used for repeated administration.

[0105] Alternatively, repeated doses of the gene therapy agent may be used. Such 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 more. Administration may continue for as long as needed, for example, at least six months, at least one year, two years, three years, four years, five years, ten years, fifteen years, twenty years, or more, up to the lifespan of the patient to be treated. The frequency of repeated doses can be determined so that the concentration of GM-CSF (especially free GM-CSF) expressed by the gene therapy agent is maintained within the therapeutic window. When repeated doses are used, the gene therapy agent can express GM-CSF as long as it is retained by the target cells. When the gene therapy agent is eliminated from the target cells (e.g., by degradation), expression of GM-CSF by the vector is stopped. Without being bound by theory, non-viral gene therapy agents, such as non-viral nucleic acid molecules including plasmids, mRNA, or self-replicating RNA molecules, typically do not integrate into the genome of target cells and are thus eliminated from the target cells over time, and therefore can be used for repeated administration. If a patient has autoimmune antibodies against GM-CSF, repeated administration may be beneficial because these antibodies will neutralize some or all of the GM-CSF expressed by a single administration of the gene therapy agent of the present invention, resulting in free GM-CSF levels lower than the therapeutic window.

[0106] Thus, the gene therapy agents of the present invention can provide repeatable and carefully controlled expression of GM-CSF (especially free GM-CSF) within a narrow therapeutic window, particularly in airway cells. Furthermore, transient expression of GM-CSF can be achieved without inducing an excessive immune response, while reducing and / or eliminating the histopathological changes in patients that are typically associated with long-term and / or high-level GM-CSF expression.

[0107] Non-viral nucleic acids The gene therapy agent of the present invention can be a non-viral nucleic acid molecule encoding GM-CSF. Typically, the non-viral nucleic acid molecule is administered using a lipid carrier as defined herein.

[0108] The nucleic acid of nucleic acid can be as defined herein.Nucleic acid can include DNA and / or RNA.Non-limiting examples of non-viral nucleic acid molecules include, as described herein, plasmid, mRNA and self-amplifying RNA (saRNA).Unless otherwise expressly indicated, all disclosures herein regarding the non-viral nucleic acid molecules of the present invention equally and without reservation apply to the plasmid, mRNA and / or saRNA molecules of the present invention.

[0109] Non-viral nucleic acid molecules can be DNA molecules or vectors, such as DNA plasmids. Non-viral nucleic acid molecules can be RNA molecules or vectors, such as mRNA vectors or self-amplifying RNA vectors. The DNA and / or RNA vectors of the present invention can be expressed in eukaryotic cells and / or prokaryotic cells. Typically, the DNA and / or RNA vectors can be expressed in the cells of the patient to be immunized, for example, the cells of mammalian or avian subjects.

[0110] The non-viral nucleic acid molecule may be a phage vector, for example the AAV / phage hybrid vector described in Hajitou et al., Cell 2006;125(2) 385-398, which is incorporated herein by reference.

[0111] Typically, in the DNA vector of the present invention, the GM-CSF transgene is operably linked to a suitable promoter as described herein. The polynucleotide may also be linked to a suitable terminator sequence. Suitable promoter and terminator sequences are well known in the art. The choice of promoter will depend on the location where the final expression of the polynucleotide will occur. Generally, constitutive promoters are preferred, but inducible promoters can also be used. The construct produced in this manner contains at least a portion of the vector, particularly the regulatory elements.

[0112] Thus, the DNA vector of the present invention typically comprises a GM-CSF transgene operably linked to a promoter. The promoter may be an inducible promoter as described herein or a non-inducible promoter. Non-limiting examples of (non-inducible) promoters are disclosed herein in the context of the plasmids of the present invention. For the avoidance of doubt, promoters disclosed in the context of a plasmid may also be operably linked to a GM-CSF transgene in any other type of DNA vector of the present invention. Furthermore, any and all disclosures herein relating to the DNA vectors (e.g., plasmids) of the present invention apply equally and without reservation to DNA vectors (e.g., plasmids) in which the GM-CSF transgene is operably linked to an inducible promoter, unless expressly indicated otherwise.

[0113] The non-viral nucleic acid molecule is preferably capable of expressing the GM-CSF transgene in a given host cell. Any suitable host cell can be used, such as mammalian, bacterial, insect, yeast, and / or plant host cells. In addition, a cell-free expression system can be used. Such expression systems and host cells are standard in the art. Typically, the non-viral nucleic acid molecule is capable of expressing the GM-CSF transgene in target cells in a patient. Non-limiting examples of suitable target cells in the lungs and airways of a patient include basal cells and submucosal gland duct cells in the upper respiratory tract, club cells and neuroendocrine cells in the bronchiolar airways, tracheal alveolar stem cells in the terminal bronchioles, type II pneumocytes, submucosal acinar cells, ionocytes, and type I pneumocytes in the alveoli.

[0114] The non-viral nucleic acid molecules of the present invention can be produced using any suitable process known in the art.Therefore, the nucleic acid molecules can be produced using chemical synthesis techniques.Alternatively, the nucleic acid molecules of the present invention can be produced using molecular biology techniques.The non-viral nucleic acid molecules of the present invention can be designed in silico and then synthesized by conventional polynucleotide synthesis techniques.

[0115] As described herein, the gene therapy agents of the present invention, including the non-viral nucleic acid molecules of the present invention, allow for careful control of the duration of expression of GM-CSF in the patient's cells and / or the concentration of GM-CSF expressed in the patient's cells, allowing for transient and / or low level expression, such that GM-CSF is expressed within a narrow toxicity / efficacy window.

[0116] Thus, the non-viral nucleic acid molecules of the invention are typically capable of transiently expressing GM-CSF, as defined herein, in a patient (i.e., in the patient's cells into which the agent is introduced). In some preferred embodiments, the transient expression of GM-CSF is for three months or less.

[0117] As described herein, expression of GM-CSF can be induced from a non-viral nucleic acid molecule of the invention a single time. The expression from a DNA vector can include transcription from an inducible or non-inducible promoter, as described herein. Expression of GM-CSF can be induced multiple times, e.g., two, three, four, five, six, seven, eight, nine, ten, or more times, from a non-viral nucleic acid molecule of the invention using an inducible promoter.

[0118] Therefore, the non-viral nucleic acid molecules of the present invention can be administered once, retained in target cells, and then used to express GM-CSF for a short period of time, as described herein.Typically, for the non-viral nucleic acid molecules of the present invention, repeated doses of the non-viral nucleic acid molecules can be used, as described herein.In particular, the frequency of repeated doses can be determined so that the concentration of GM-CSF (particularly free GM-CSF) expressed by the non-viral nucleic acid molecules of the present invention is maintained within the therapeutic window.

[0119] The non-viral nucleic acid molecule of the present invention may be codon-optimized for expression in a specific cell type, for example, eukaryotic cells (e.g., mammalian cells, yeast cells, insect cells, or plant cells) or prokaryotic cells (e.g., E. coli). The term "codon-optimized" refers to the replacement of at least one codon in the basic polynucleotide sequence with the codon that is preferentially used by the host organism in which the polynucleotide is to be expressed. Typically, the codon that is most frequently used in the host organism is used in the codon-optimized polynucleotide sequence. The method of codon optimization is well known in the art.

[0120] Those skilled in the art will understand that many different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code. Furthermore, those skilled in the art will also understand that, using routine techniques, nucleotide substitutions can be made to reflect the codon usage of any particular host organism in which the polypeptide is to be expressed, without affecting the polypeptide sequence encoded by the nucleic acid molecule. Thus, unless otherwise indicated, nucleic acids encoding GM-CSF according to the present invention include all polynucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence.

[0121] The DNA molecules of the present invention typically comprise a promoter operably linked to a nucleic acid sequence encoding GM-CSF, where operably linked means that the promoter is configured to express the nucleic acid sequence encoding the signal peptide and / or the nucleic acid sequence encoding GM-CSF.

[0122] The (non-viral) nucleic acid molecule of the present invention may comprise at least a portion of a vector, particularly a regulatory element. As a non-limiting example, a promoter within a DNA molecule of the present invention can be used to express more than one polypeptide, including one or more therapeutic proteins in addition to GM-CSF. Thus, a DNA molecule of the present invention may contain a nucleic acid sequence that, when transcribed, produces multiple polypeptides; for example, a transcript may contain multiple open reading frames (ORFs) and may also contain one or more internal ribosome entry sites (IRES) to enable translation of ORFs after the first ORF. A transcript may be polycistronic, i.e., it may be transcribed to produce a polypeptide, which may then be cleaved to yield multiple polypeptides. Alternatively, a DNA molecule of the present invention may contain multiple promoters and thus produce multiple transcripts, thus producing multiple polypeptides, including multiple therapeutic proteins, including GM-CSF. A nucleic acid may, for example, express one, two, three, four, or more polypeptides using one or more promoters.

[0123] The (non-viral) nucleic acid molecule of the present invention may contain one or more translation initiation sequences (TIS). Translation initiation plays an important role in mRNA translation, typically by initiation-specific methionyl-tRNA (Met-tRNAi) identifying the AUG start codon and triggering the downstream translation process. Non-canonical start codons (e.g., CUG for valyl-tRNA) / TISs can also be used.

[0124] The DNA molecules of the present invention may contain at least one termination signal. A "termination signal" or "terminator" is composed of a DNA sequence involved in the specific termination of an RNA transcript by an RNA polymerase. Thus, termination signals that terminate the production of an RNA transcript are contemplated by the present invention. Terminators are required in vivo to achieve desired message levels. In eukaryotic systems, terminator regions may also contain specific DNA sequences that enable site-specific cleavage of the new transcript to expose a polyadenylation site. This signals specialized endogenous polymerases to add a stretch of approximately 200 A residues (polyA) to the 3' end of the transcript. RNA molecules modified with this polyA tail are believed to be more stable and translated more efficiently. Therefore, when a nucleic acid is intended for expression in eukaryotes, the terminator typically contains a signal for RNA cleavage, and it is preferred that the terminator signal promotes message polyadenylation. Terminator and / or polyadenylation site elements may function to enhance message levels and minimize readthrough from the cassette into other sequences.

[0125] Terminators contemplated for use in the present invention include any known transcription terminators described herein or known to those skilled in the art, including, but not limited to, gene termination sequences, such as the bovine growth hormone terminator, or viral termination sequences, such as the SV40 terminator. In certain embodiments, the termination signal may lack a transcribable or translatable sequence, such as that resulting from sequence truncation.

[0126] Non-viral nucleic acid molecules (e.g., plasmids) of the invention, or portions thereof, may be codon-optimized. As a non-limiting example, the GM-CSF transgene may be codon-optimized, and / or the promoter may be codon-optimized, or the entire molecule may be codon-optimized.

[0127] The non-viral nucleic acid molecules (e.g., plasmids) of the present invention or portions thereof may be modified to reduce their CpG dinucleotide content. Thus, the non-viral nucleic acid molecules (e.g., plasmids) of the present invention or portions thereof may have a low CpG dinucleotide content or no CpG dinucleotide content at all. A low CpG content means 20 or less, 15 or less, 10 or less, or 5 or less CpG dinucleotides (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 CpG dinucleotides). In the non-viral nucleic acid molecules (e.g., plasmids) of the present invention or portions thereof, all CG dinucleotides are replaced with any one of AG, TG, or GT. Thus, the non-viral nucleic acid molecules (e.g., plasmids) of the present invention or portions thereof may not contain CpG.

[0128] The GM-CSF transgene in the non-viral nucleic acid molecules (e.g., plasmids) of the present invention may have a low CpG dinucleotide content as defined herein, preferably the GM-CSF transgene contains 10 or fewer CpG dinucleotides or no CpG dinucleotides. Alternatively, or in addition, the promoter in the non-viral nucleic acid molecules (e.g., plasmids) of the present invention may have a low CpG dinucleotide content as defined herein, preferably the promoter contains 10 or fewer CpG dinucleotides or no CpG dinucleotides. Preferably, both the GM-CSF transgene and promoter in the non-viral nucleic acid molecules (e.g., plasmids) of the present invention have a low CpG dinucleotide content as defined herein, preferably the GM-CSF transgene and promoter each contain 10 or fewer CpG dinucleotides or no CpG dinucleotides.

[0129] The nucleic acid of the present invention can be used in the production of retroviral / lentiviral (e.g., SIV) vectors as described herein.By way of non-limiting example, the non-viral nucleic acid of the present invention can be a plasmid, which can be used in the treatment of PAP as described herein, or can be used in the production of viral / retroviral / lentiviral (e.g., SIV) vectors of the present invention.The nucleic acid of the present invention is contained in viral / retroviral / lentiviral (e.g., SIV) vectors.

[0130] Typically, the non-viral nucleic acids of the invention are capable of expressing a therapeutic protein in airway cells (as described herein).

[0131] Non-viral nucleic acid molecules are unable to replicate in the subject to be treated because they lack the viral genetic material to hijack the body's normal production mechanisms, but they are capable of replicating in suitable host cells, such as yeast or bacteria, including E. coli, and particularly airway cells as defined herein.

[0132] Plasmid The term "plasmid," as used herein, refers to a construct composed of genetic material designed to induce transformation of a targeted cell. A plasmid comprises a plasmid backbone. A "plasmid backbone," as used herein, comprises multiple genetic elements oriented in a position and order with other necessary genetic elements so that the nucleic acid in the nucleic acid is transcribed and, if necessary, translated in the transfected cell.

[0133] The plasmid backbone may contain one or more unique restriction sites within the backbone. The plasmid may be capable of autonomous replication in a given host or organism, allowing the cloned sequences to be reproduced. The plasmid may confer some well-defined phenotype on the host organism, which is selectable or easily detected. The plasmid or plasmid backbone may have a linear or circular configuration. Components of the plasmid may include, but are not limited to, (1) the plasmid backbone; (2) a sequence encoding a signal peptide; (3) a sequence encoding GM-CSF and optionally one or more additional therapeutic proteins; and (4) a DNA molecule incorporating regulatory elements for transcription, translation, RNA stability, and replication.

[0134] The purpose of a plasmid in human gene therapy is to efficiently deliver a nucleic acid sequence to cells or tissues and express a therapeutic protein therein. Specifically, the purpose of a plasmid is to achieve high copy number, avoid potential sources of plasmid instability, and provide a means for plasmid selection. With regard to expression, the nucleic acid of the present invention contains the elements necessary for expression of the GM-CSF transgene contained in the nucleic acid. Expression includes efficient transcription of the inserted gene, nucleic acid sequence, or nucleic acid within the plasmid.

[0135] Thus, the plasmids of the present invention typically comprise a GM-CSF transgene operably linked to a promoter. The promoter may be an inducible promoter as described herein. Any and all disclosures herein regarding the plasmids of the present invention apply equally and without reservation to plasmids in which the GM-CSF transgene is operably linked to an inducible promoter, unless expressly indicated otherwise.

[0136] Alternatively, the promoter may be a (non-inducible) promoter capable of expressing GM-CSF in one or more target cell types. Non-limiting examples of suitable target cells within the lungs and airways of a patient include basal cells and submucosal gland duct cells in the upper respiratory tract, club cells and neuroendocrine cells in the bronchiolar airways, tracheal alveolar stem cells in the terminal bronchioles, type II pneumocytes, submucosal acinar cells, ionocytes, and type I pneumocytes in the alveoli.

[0137] Non-limiting examples of promoters that can be used according to the present invention, particularly those that may be operably linked to a GM-CSF transgene in a non-viral nucleic acid molecule (e.g., a plasmid) of the present invention, include a hybrid human CMV enhancer / EF1a (hCEF) promoter, a cytomegalovirus (CMV) promoter, and an elongation factor 1a (EF1a) promoter. Preferably, the non-viral nucleic acid molecule (e.g., a plasmid) comprises a hybrid human CMV enhancer / EF1a (hCEF) promoter.

[0138] A preferred example of the hCEF promoter sequence of the present invention is provided by SEQ ID NO: 6. Alternatively, the promoter may be a CMV promoter. An example of a CMV promoter sequence is provided by SEQ ID NO: 26 or 7, preferably SEQ ID NO: 26. The promoter may also be a human elongation factor 1a (EF1a) promoter. An example of an EF1a promoter is provided by SEQ ID NO: 8. Other promoters for transgene expression are known in the art, and their suitability for the non-viral nucleic acid molecules (e.g., plasmids) of the present invention is determined using routine techniques known in the art. Non-limiting examples of other promoters include UBC and UCOE. As described herein, promoters may be modified to further regulate the expression of the transgenes of the present invention.

[0139] The promoter contained in the non-viral nucleic acid molecule (e.g., plasmid) of the present invention may be specifically selected and / or modified to further refine the regulation of the expression of the GM-CSF gene. Again, suitable promoters and standard techniques for their modification are known in the art. As a non-limiting example, some suitable (CpG-free) promoters suitable for use in the present invention are described in Pringle et al. (J. Mol. Med. Berl. 2012, 90(12):1487-96), which is incorporated herein by reference in its entirety.

[0140] Plasmids of the invention or portions thereof may be codon optimized. By way of non-limiting example, the GM-CSF transgene may be codon optimized and / or the promoter may be codon optimized, or the entire plasmid may be codon optimized.

[0141] The plasmid of the present invention or a portion thereof may be modified to reduce its CpG dinucleotide content. Thus, the plasmid of the present invention or a portion thereof may have a low CpG dinucleotide content or no CpG dinucleotide content at all. A low CpG content means 20 or less, 15 or less, 10 or less, or 5 or less CpG dinucleotides (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 CpG dinucleotides). In the plasmid of the present invention or a portion thereof, all CG dinucleotides are replaced with any one of AG, TG, or GT. Thus, the plasmid of the present invention or a portion thereof may not contain CpG.

[0142] The GM-CSF transgene in the plasmid of the present invention may have a low CpG dinucleotide content as defined herein, preferably the GM-CSF transgene contains 10 or fewer CpG dinucleotides or no CpG dinucleotides. Alternatively, or in addition, the promoter in the plasmid of the present invention may have a low CpG dinucleotide content as defined herein, preferably the promoter contains 10 or fewer CpG dinucleotides or no CpG dinucleotides. Preferably, both the GM-CSF transgene and promoter in the plasmid of the present invention have a low CpG dinucleotide content as defined herein, preferably the GM-CSF transgene and promoter each contain 10 or fewer CpG dinucleotides or no CpG dinucleotides.

[0143] Preferably, the non-viral nucleic acid molecule (e.g., a plasmid) of the present invention comprises an hCEF promoter with low or no CpG dinucleotide content. Low CpG content means 20 or less, 15 or less, 10 or less, or 5 or less CpG dinucleotides (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 CpG dinucleotides). In the hCEF promoter, all CG dinucleotides are replaced with any one of AG, TG, or GT. Therefore, the hCEF promoter may not contain CpG. A preferred example of a CpG-free hCEF promoter sequence of the present invention is provided by SEQ ID NO: 6. The absence of CpG dinucleotides typically further improves the performance of the non-viral nucleic acid molecules (e.g., plasmids) of the present invention, particularly in situations where it is undesirable to induce an immune response to the expressed antigen or an inflammatory response to the delivered expression construct. The elimination of CpG dinucleotides reduces the occurrence of flu-like symptoms and inflammation that can result from administration of the construct, particularly when administered to the respiratory tract.

[0144] The non-viral nucleic acid molecules (e.g., plasmids) of the present invention may be modified to allow gene expression to be silenced. Standard techniques for modifying vectors in this manner are known in the art. As a non-limiting example, Tet-responsive promoters are widely used.

[0145] The non-viral nucleic acid molecules (eg, plasmids) of the invention may be codon optimized as described herein.

[0146] Methods for preparing plasmid DNA are well known in the art. Typically, they are capable of autonomous replication in a suitable host or producer cell.

[0147] The host cell containing the plasmid (e.g., transformed, transfected, or electroporated) can be of prokaryotic or eukaryotic nature, with the plasmid stably or transiently transformed, transfected, or electroporated. Suitable host cells include bacteria, yeast, fungi, invertebrate, and mammalian cells. Preferably, the host cell is a bacterium; more preferably, it is E. coli.

[0148] The host cells can then be used in methods for large-scale production of the plasmid. The cells are grown in a suitable culture medium under favorable conditions, and the desired plasmid is isolated from the cells, or from the medium in which the cells were grown, by any purification technique known to those skilled in the art; see, e.g., Sambrook et al. (supra).

[0149] The present invention also provides a host cell comprising a nucleic acid (e.g., a plasmid) of the present invention. Typically, the host cell is a mammalian cell, particularly a human cell or cell line. Non-limiting examples of host cells include HEK293 cells (e.g., HEK293F or HEK293T cells) and 293T / 17 cells. Commercially available cell lines suitable for virus production are also readily available (described herein).

[0150] Non-limiting examples of plasmids according to the present invention include pIC017 hCEF GMCSF, which is depicted in FIG. 1A, and pIC098 CMV GMCSF, which is depicted in FIG. 1B.

[0151] pIC017 hCEF GMCSF (shown in Figure 1A) contains a GM-CSF transgene under the control of the hCEF promoter, a bovine growth hormone (BGM) polyA sequence, an R6K origin of replication (containing CpG dinucleotides), and a kanamycin resistance cassette (also containing CpG dinucleotides). Additionally, the pIC017 plasmid contains a chimeric intron downstream of the enhancer / promoter region. The chimeric intron consists of a 5' donor site derived from the first intron of the human β-globin gene and a branch and 3' acceptor site derived from an intron located between the leader and body of the immunoglobulin gene heavy chain variable region. The sequences of the donor and acceptor sites, along with the branch point site, have been altered to match consensus sequences for splicing. The presence of introns flanking the transgene, particularly in pIC017, has been shown to increase the level of gene expression. An exemplary β-globin / IgG chimeric intron sequence is provided in SEQ ID NO:9. pIC017 hCEF GMCSF has the nucleic acid sequence of SEQ ID NO: 10. Variants thereof (described herein) are also included, particularly variants having at least 90% (e.g., at least 90, 92, 94, 95, 96, 97, 98, 99, or 100%) similarity to SEQ ID NO: 10. Elements of pIC017 hCEF GMCSF can be replaced to provide additional exemplary plasmids of the invention. As non-limiting examples, the mouse GM-CSF transgene can be replaced with a human GM-CSF transgene, such as that of SEQ ID NO: 2 described herein, the hCEF promoter can be replaced with another promoter, preferably an inducible promoter described herein, and / or CpG dinucleotides can be removed from one or more elements of the pIC017 hCEF GMCSF plasmid.

[0152] pIC098 CMV GMCSF (diagrammed in FIG. 1B) contains a GM-CSF transgene under the control of a CMV promoter, a bovine growth hormone (BGM) polyA sequence, an R6K origin of replication (containing CpG dinucleotides), and a kanamycin resistance cassette (also containing CpG dinucleotides). pIC098 also contains a β-globin / IgG chimeric intron, as described above in the context of pIC017. pIC098 CMV GMCSF has the nucleic acid sequence of SEQ ID NO: 11. Variants thereof (as described herein) are also included, particularly variants having at least 90% (e.g., at least 90, 92, 94, 95, 96, 97, 98, 99, or 100%) similarity to SEQ ID NO: 11. Elements of pIC098 CMV GMCSF can be substituted to provide additional exemplary plasmids of the invention. As non-limiting examples, the mouse GM-CSF transgene may be replaced with a human GM-CSF transgene, such as that of SEQ ID NO: 2 described herein, the CMV promoter may be replaced with another promoter, preferably an inducible promoter described herein, and / or CpG dinucleotides may be removed from one or more elements of the pIC098 CMV GMCSF plasmid.

[0153] mRNA and saRNA The non-viral nucleic acid molecule of the present invention can be mRNA or self-amplifying RNA (saRNA) encoding GM-CSF. Both mRNA and saRNA can be transfected into target cells. Once inside target cells, mRNA or saRNA is translated by host cells, resulting in the production of GM-CSF protein. Typically, mRNA and / or saRNA molecules are linear RNA molecules.

[0154] In addition to the nucleic acid sequence encoding GM-CSF, the mRNA of the present invention typically contains the following basic elements: (i) a cap; (ii) a 5' UTR; (iii) a 3' UTR; and (iv) a poly(A) tail (which may be of variable length). These elements may be as defined herein. The mRNA of the present invention typically has a sequence length of about 0.2 kb to about 10 kb, e.g., about 0.2 kb to about 7 kb, about 0.2 kb to about 5 kb, about 0.5 kb to about 5 kb, or about 0.5 kb to about 2 kb, with a sequence length of about 0.5 kb to about 5 kb or about 0.5 kb to about 2 kb being preferred.

[0155] saRNA is a type of RNA molecule that shares many structural similarities with mRNA: it is a linear, single-stranded RNA molecule that shares elements with mRNA. Specifically, in addition to the nucleic acid sequence encoding GM-CSF, the saRNA of the present invention typically contains the following basic elements: (i) a cap; (ii) a 5' untranslated region (UTR, also referred to as a conserved sequence element or CSE); (iii) alphavirus nonstructural proteins 1-4 (nsP1-4) encoding the replicase described herein; (iv) a subgenomic promoter and / or internal ribosome entry site (IRES); (v) a 3' UTR (or CSE); and (vi) a poly(A) tail (which may be of variable length). These elements may be as defined herein. The main difference between saRNA and mRNA is that saRNA is typically longer than mRNA. The saRNA of the present invention typically has a sequence length of about 8 kb to about 15 kb, for example, about 8 kb to about 12 kb, about 9 kb to about 12 kb, or about 9 kb to about 10 kb, with a sequence length of about 9 kb to about 12 kb or about 9 kb to about 10 kb being preferred.

[0156] The difference in size between saRNA and mRNA is due to the fact that saRNA typically encodes at least one protein (e.g., one, two, three, or four additional proteins) in addition to GM-CSF. Specifically, saRNA typically encodes at least a replicase in addition to GM-CSF. Typically, saRNA encodes four additional proteins in addition to GM-CSF. The four additional proteins encode an RNA-dependent RNA polymerase (RdRP) complex, which amplifies the synthetic transcript in situ and results in efficient expression of the GM-CSF protein in target cells. Therefore, a lower dose / concentration of saRNA may be required to treat a patient compared to an equivalent mRNA (or plasmid).

[0157] The backbone sequence of the saRNA, including the gene encoding the RdRP complex, is typically derived from an alphavirus, such as Venezuelan equine encephalitis virus (VEEV), Sindbis virus (SINV), and Semliki Forest virus (SFV), preferably VEEV.

[0158] In the saRNA of the present invention, the sequence encoding GM-CSF is downstream of a subgenomic promoter and / or an IRES.

[0159] The mRNA and / or saRNA of the present invention can be synthesized as unmodified or modified mRNA. Typically, the mRNA and / or saRNA can contain one or more chemical and structural modifications to reduce immunogenicity and improve mRNA stability, such as inhibiting mRNA interaction with toll-like receptors TLR3, TLR7, TLR8, and retinoid-inducible gene I (RIG-I). Therefore, the mRNA or saRNA molecules of the present invention are typically modified to replace any uridine base with the chemically modified alternative pseudouridine (ψ or ψ-UTP). The use of pseudouridine is well known in the art. Alternatively, or in addition, any cytidine base can be replaced with the chemically modified alternative 5-methylcytidine (m5C), which is also well known in the art. Substitution of uridine with pseudouridine and / or cytidine with 5-methylcytidine typically reduces the degradation of mRNA and / or saRNA by target cells, allowing for enhanced translation of mRNA and / or saRNA molecules and increased GM-CSF protein expression. Other chemically modified bases may be used alone or in combination. Non-limiting examples of such bases include m6A, 5-methyluridine (m5U), 2-thiouridine (s2U), and / or N1-methylpseudouridine (N1-m ψ-UTP), with N1-m ψ-UTP being particularly preferred.

[0160] Other modifications to the mRNA and / or saRNA may be made instead of, or in addition to, the chemical modification of one or more bases described above. Any combination of the modifications described herein may be used.

[0161] mRNA and / or saRNA can comprise RNA backbone modification.Usually, backbone modification is the modification that chemically modifies the phosphate of the backbone of the nucleotide contained in RNA.Exemplary backbone modification typically includes but is not limited to the modification from the group consisting of methyl phosphonate, methyl phosphoramidate, phosphoramidate, phosphorothioate (for example, cytidine 5'-O-(1-thiophosphate)), boranophosphate, positively charged guanidinium group, etc., which means that phosphodiester linkage is replaced with other anionic, cationic or neutral group.

[0162] The mRNA and / or saRNA may contain sugar modifications. Typical sugar modifications are chemical modifications of the sugar of the nucleotide, including 2'-deoxy-2'-fluoro-oligoribonucleotide (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamine-oligoribonucleotide (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O-alkyl oligoribonucleotide, 2'-deoxy-2'-C- Sugar modifications include, but are not limited to, alkyl oligoribonucleotides (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyl oligoribonucleotides, and their isomers (2'-aracytidine 5'-triphosphate, 2'-arauidine 5'-triphosphate), or azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0163] Stabilizing modifications can be made to one or both of the 3' and 5' ends of the mRNA and / or saRNA. Preferably, the stabilizing modifications are made to the 5' end, and optionally also to the 3' end. Non-limiting examples of stabilizing modifications include, for example, end-capping, polyA tailing, replacement of unstable non-coding sequences (e.g., adenylate-uridylate-rich elements (AREs)), or addition of 3' or 5' untranslated sequences derived from stable mRNAs (e.g., β-globin, actin, GAPDH, tubulin, histones, or citric acid cycle enzyme mRNAs). Stabilizing modifications can also be made within the mRNA and / or saRNA, including, for example, codon optimization and / or modification of Kozak sequences and / or incorporation of modified nucleosides (e.g., pyrrolo-pyrimidine, C5-iodouridine, 2-aminoadenosine, 2-thiothymidine, etc.).

[0164] Typically, the mRNA and / or saRNA of the present invention comprise a cap. The presence of a cap is important for providing resistance to nucleases found in most eukaryotic cells. 5'-capping typically stabilizes the mRNA and / or saRNA, helping the molecule evade the patient's immune system. Thus, in some embodiments, the mRNA and / or saRNA of the present invention comprise a 5'-cap structure. The 5'-cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphate by a guanylyltransferase, generating a 5'5'5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. The 5'-cap is not particularly limited, and examples of 5'-caps are known in the art. As a non-limiting example, a 5'-cap can be added using a Cap1 ( m7 GpppG 2’Om N), Cap2( m7 GpppN 2’Om N 2’Om), m 7 GpppG analog, anti-reverse cap analog (ARCA, anti-reverse cap analog; m2 7,3’-O G(5')ppp(5')A, G(5')ppp(5')A, and G(5')ppp(5')G. 5' Cap1 may be preferred because it mimics the natural eukaryotic mRNA structure and 2' O-methylation may reduce recognition of mRNA and / or saRNA by pattern recognition receptors.

[0165] The mRNA and / or saRNA of the present invention may contain 5' and / or 3' untranslated regions (UTRs). The 5' and / or 3' UTRs, particularly the 5' UTR, may contain one or more elements that improve the nuclease resistance and / or half-life of the mRNA and / or saRNA, such as an iron-responsive element. The 5' UTR may be approximately 50 to 500 nucleotides in length. The 3' UTR may contain one or more of the following: a polyadenylation signal (e.g., a poly(A) tail described herein), a binding site for a protein that affects the stability and / or subcellular location of the mRNA and / or saRNA, and / or one or more binding sites for miRNA. The 3' UTR may be 50 to 500 nucleotides in length, or may be longer. In the saRNA of the present invention, the 5' and / or 3' UTR may contain a conserved sequence element (CSE). CSEs are present in alphavirus genomes and are believed to bind to viral and / or cellular proteins and regulate viral RNA synthesis.

[0166] Typically, the mRNA and / or saRNA of the present invention contain a tail. The presence of the "tail" functions to protect the mRNA and / or saRNA from exonuclease degradation, thus increasing the half-life of the mRNA and / or saRNA. Therefore, the mRNA and / or saRNA of the present invention may contain a 3' poly(A) tail structure. The poly(A) tail on the 3' end of the mRNA and / or saRNA typically contains about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 90 adenosine nucleotides, about 20 to 80 adenosine nucleotides, or about 120 to 150 adenosine nucleotides, preferably about 80 adenosine nucleotides). In some embodiments, the mRNA of the present invention contains a 3' poly(C) tail structure. Alternatively, or in addition, the mRNA and / or saRNA of the present invention may contain a 3' poly(C) tail structure. A suitable poly(C) tail on the 3' end of an mRNA typically contains about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). The poly(C) tail may be in addition to or replace the poly(A) tail.

[0167] In some preferred embodiments, the non-viral nucleic acid molecule of the present invention is an mRNA or saRNA that contains a sequence encoding GM-CSF and one or more of: (i) a uridine replaced by a pseudouridine; (ii) a 5' Cap1; and / or (ii) a poly(A) tail of about 10-100 adenosine nucleotides, preferably about 80 adenosine nucleotides. Particularly preferred are mRNA and / or saRNA molecules that contain all of (i) through (iii).

[0168] The mRNA and / or saRNA of the present invention are typically synthetic molecules structurally similar to natural mRNA counterparts, and when transfected into target cells, rapidly express GM-CSF protein. The mRNA and / or saRNA of the present invention can be synthesized by any of a variety of known methods. For example, the mRNA and / or saRNA of the present invention can be synthesized by in vitro transcription (IVT). Briefly, IVT is typically carried out using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may contain DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor. The exact conditions can be easily determined by those skilled in the art.

[0169] Lipid Carrier The gene therapy agents of the present invention may contain a lipid carrier to facilitate delivery to a patient and / or uptake by target cells. Typically, when the gene therapy agent of the present invention contains a non-viral nucleic acid molecule, the agent also further contains a lipid carrier.

[0170] Lipid carriers can be formulated as lipid nanoparticles.The terms " lipid nanoparticles ", " lipid carrier vehicle " and " lipid-derived nanoparticles " are all used interchangeably and refer to a delivery vehicle that comprises one or more lipids (e.g., cationic lipids, non-cationic lipids, cholesterol-based lipids, and PEG-modified lipids).The contemplated lipid nanoparticles can be prepared by including a multi-component lipid mixture with various ratios using one or more cationic lipids, non-cationic lipids, cholesterol-based lipids, and PEG-modified lipids.Examples of suitable lipids include, for example, phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides). Preferably, the lipid nanoparticle is a liposome, which is a bilayer vesicle typically formed by amphiphilic molecules, such as synthetic or naturally occurring lipids, containing spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16: 307-321, 1998). The bilayer membrane of the liposome can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.).

[0171] In the context of the present invention, lipid carrier vehicles typically function to transport the non-viral nucleic acid molecules of the present invention into target cells. For the purposes of the present invention, liposome transfer vehicles are prepared to contain the desired nucleic acid. The process of incorporating a desired entity (e.g., a non-viral nucleic acid molecule) into liposomes is often referred to as "loading" (Lasic et al., FEBS Lett., 312:255-258, 1992). The nucleic acid incorporated into liposomes is located completely or partially in the internal space of the liposome, within the bilayer membrane of the liposome, or associated with the outer surface of the liposome membrane. The incorporation of nucleic acid into liposomes is also referred to herein as "encapsulation," in which the nucleic acid is completely contained within the internal space of the liposome. The purpose of incorporating the non-viral nucleic acid molecules of the present invention into transfer vehicles, such as liposomes, is often to protect the nucleic acid from environments that may contain enzymes or chemicals that degrade the nucleic acid and / or systems or receptors that cause the nucleic acid to be rapidly excreted. Thus, in some embodiments of the present invention, the transfer vehicle selected may enhance the stability of the non-viral nucleic acid molecules of the present invention contained therein. Liposomes may enable the encapsulated non-viral nucleic acid molecules of the present invention to reach target cells.

[0172] As used herein, liposome delivery vehicles are generally characterized as microscopic vesicles having an internal aqueous space separated from the outside medium by one or more bilayer membranes.

[0173] Suitable lipid carriers can comprise cationic lipids.As used herein, the term "cationic lipid" refers to any of several lipid species that have a net positive charge at selected pH, for example, physiological pH.A number of cationic lipids have been described in the literature, and many of them are commercially available.In certain embodiments, the composition of the present invention comprises the ionizable cationic lipids described in US Provisional Patent Application No. 61 / 617,468 filed on March 29, 2012, such as (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine (HGT5000), ... Lipid nanoparticles including (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine (HGT5001), and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (HGT5002), and the like, may be utilized.

[0174] Any suitable delivery means can be used to deliver the gene therapy agent of the present invention, particularly the non-viral nucleic acid molecule of the present invention (for example, plasmid, mRNA or saRNA) to target cell or patient.Suitable delivery means are known in the art and are within the ordinary skill of those skilled in the art.Non-limiting examples include the use of cationic lipids, polymers (for example, polyethyleneimine and poly-L-lysine) and electroporation.

[0175] Typically, lipid carriers according to the present invention comprise one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids.

[0176] Preferably, lipid carriers comprising one or more cationic lipids are used to deliver the non-viral nucleic acid molecules of the present invention (such as plasmid, mRNA or saRNA) to target cells or patients.Non-limiting examples of cationic lipids suitable for use according to the present invention include GL67A and lipofectamine.Further non-limiting examples of lipid carriers include C12-200, HGT4003, HGT5000, HGT5001, ICE, DLinKC2-DMA, DODAP, DODMA, DLinDMA and CLinDMA, which are described in European Patent No. EP2858679B1, which is incorporated herein by reference in its entirety.

[0177] The non-cationic lipid that can be contained in the lipid carrier of the present invention can be defined as neutral lipid, that is, lipid that has no net charge under the condition that the composition is formulated and / or administered.Non-limiting examples of non-cationic lipid include DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)) and cholesterol.

[0178] Non-limiting examples of cholesterol-based lipids that can be included in the lipid carriers of the present invention include DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol) and 1,4-bis(3-N-oleylamino-propyl)piperazine.

[0179] Non-limiting examples of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids that can be included in the lipid carriers of the present invention include derivatized ceramides (PEG-CER), such as N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)-2000] (C8 PEG-2000 ceramide). PEG-modified lipids can include, but are not limited to, polyethylene glycol chains up to 5 kDa in length covalently attached to lipids having alkyl chains of C6 to C20 in length. The addition of such moieties can prevent complex aggregation and also provide a means for increasing the circulatory lifetime of lipid-nucleic acid compositions and their delivery to target cells (Klibanov et al. (1990) FEBS Letters, 268(1):235-237), or they may be selected for rapid release from the formulation in vivo (see U.S. Pat. No. 5,885,613).

[0180] Preferably, the lipid carrier is GL67A. The cationic lipid mixture GL67A is a mixture of three components: GL67 (cholest-5-en-3-ol (3β)-, 3-[(3-aminopropyl) [4-[(3-aminopropyl) amino] butyl] carbamate] (CAS number: 179075-30-0)), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), and DMPE-PEG5000 (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)5000]). These components are formulated in a molar ratio of 1:2:0.05 to form GL67A. The composition of GL67A and the method for its production are disclosed in International Publication No. WO2013 / 061091, which also discloses a method for preparing a mixture of GL67A and an exemplary non-viral vector. The contents of International Publication No. WO2013 / 061091 are incorporated herein by reference in their entirety.

[0181] Lipofectamine consists of a 3:1 mixture of DOSPA (2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propaniminium trifluoroacetate) and DOPE.

[0182] The lipid carriers of the present invention, particularly GL67A, can be used at lipid:non-viral nucleic acid molecule ratios of about 1:1 to about 7:1, preferably about 2:1 to about 6:1, and more preferably about 2:1 to about 4:1. Exemplary ratios include 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, and 7:1, preferably 2:1, 3:1, or 4:1. The non-viral nucleic acid molecule can be RNA (particularly mRNA) or a plasmid, as described herein.

[0183] viral vectors The gene therapy agent of the present invention can be a viral vector. Thus, as described herein, a viral vector can be used to transiently express GM-CSF in a patient to treat PAP.

[0184] The viral vectors of the present invention comprise an inducible promoter as described herein. The inclusion of an inducible promoter in the viral vectors of the present invention allows for careful control of the concentration of GM-CSF expressed in a patient's cells, allowing for transient and / or low-level expression, resulting in GM-CSF being expressed within a narrow toxicity / efficacy window. In this way, the viral vectors of the present invention allow for the treatment of PAP, particularly aPAP, while simultaneously reducing or eliminating the side effects associated with overexpression of GM-CSF in the lungs.

[0185] The viral vector of the present invention can be a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus (AAV) vector, a baculoviral vector, a herpes simplex virus (HSV) vector, or a poxvirus vector.As detailed herein, retroviral vectors and lentiviral vectors are preferred, and lentiviral vectors are particularly preferred.

[0186] The viral vectors of the present invention enable the expression of therapeutic levels of GM-CSF. The viral vectors of the present invention typically provide therapeutic levels of GM-CSF expression when administered to a patient. Expression can be measured by any appropriate method (qualitative or quantitative, preferably quantitative), and concentrations are provided in any appropriate units of measurement, such as ng / ml or nM. As described herein, therapeutic levels or concentrations of GM-CSF expression are relatively low, and may even be below the lower limit of detection when quantifying GM-CSF levels using standard assays, such as bronchoalveolar lavage fluid (BALF) or lung tissue. However, therapeutic effects can still be quantified based on parameters such as BALF turbidity, surfactant protein D (SF-D) concentration in BALF or lungs, surfactant deposition in the lungs, CT scans, and / or pulmonary function metrics, as described herein.

[0187] Viral vectors are typically non-replicating or replication-impaired, meaning that they are unable to replicate to any significant extent in normal cells (e.g., normal human cells) as measured by conventional means, such as measuring DNA synthesis and / or viral titer. Non-replicating or replication-impaired vectors may be naturally occurring (i.e., isolated from nature) or artificially occurring (e.g., by in vitro propagation or genetic engineering). Generally, there is at least one cell type in which replication-impaired viral vectors can grow; for example, the poxvirus vector modified vaccinia Ankara (MVA) can be grown in CEF cells. In one embodiment, the vector is selected from a human or simian adenovirus or poxvirus vector.

[0188] Typically, viral vectors are incapable of causing significant infection in an animal subject, typically a mammalian subject, such as a human or other primate.

[0189] The nucleic acid sequence encoding GM-CSF to be included in the viral vector of the present invention may be modified to enhance expression. For example, the GM-CSF transgene sequence may be CpG-deleted (or CpG-free) and / or codon-optimized to enhance gene expression. Standard techniques for modifying transgene sequences in this manner are known in the art. The promoter in the viral vector may be CpG-deleted (or CpG-free) and / or codon-optimized. The genome of the viral vector may have a low CpG dinucleotide content or may be free of CpG dinucleotides (the above disclosure regarding codon optimization and / or CpG deletion for non-viral nucleic acid molecules applies equally and without reservation to the viral vector of the present invention).

[0190] The viral vectors of the invention can be used to transduce isolated and expanded stem / progenitor cells ex vivo prior to administration to a patient. Preferably, the viral vectors of the invention are used to transduce cells within the lung (or airway / respiratory tract) in vivo.

[0191] The present invention also provides a host cell comprising the viral vector of the present invention. Typically, the host cell is a mammalian cell, particularly a human cell or cell line. Non-limiting examples of host cells include HEK293 cells (e.g., HEK293F or HEK293T cells) and 293T / 17 cells. Commercially available cell lines suitable for virus production are also readily available (described herein).

[0192] Retroviral and lentiviral vectors The gene therapy agents of the present invention can be retroviral or lentiviral vectors. Thus, as described herein, retroviral / lentiviral vectors can be used to transiently express GM-CSF in patients to treat PAP.

[0193] The retroviral / lentiviral vector of the present invention is integrated into the genome of the transduced cell. In the context of the present invention, the integration of the retroviral / lentiviral vector into the genome of the target cell has the ability to allow transient expression of GM-CSF to be induced for a long period of time, rather than continuous long-term expression. This is because, once integrated, the inducible promoter is transiently induced to drive GM-CSF expression according to the patient's clinical needs (described herein), and this induction / transient expression is repeated according to the patient's clinical needs over a long period of time.

[0194] The retroviral / lentiviral vectors of the present invention comprise an inducible promoter as described herein. By including an inducible promoter in the retroviral / lentiviral vectors of the present invention, the concentration of GM-CSF (particularly free GM-CSF) expressed in the patient's cells can be carefully controlled, allowing for transient and / or low-level expression, resulting in GM-CSF (particularly free GM-CSF) being expressed within a narrow toxicity / efficacy window. In this way, the retroviral / lentiviral vectors of the present invention enable the treatment of PAP, particularly aPAP, while simultaneously reducing or eliminating the side effects associated with overexpression of GM-CSF in the lungs. Alternatively, a promoter that provides transient GM-CSF expression may be selected. As a non-limiting example, the inventors have demonstrated a CMV promoter (or a CMV promoter and enhancer) that drives expression for less than 22 days. Thus, unlike viral gene therapy agents for other indications where long-term expression is desired, the present invention may involve retroviral / lentiviral vectors in which GM-CSF expression is under the control of a CMV promoter (or a CMV promoter and enhancer).

[0195] The term "retrovirus" refers to any member of the retrovirus family of RNA viruses that encode reverse transcriptase. The term "lentivirus" refers to a family of retroviruses. Thus, all references herein to the retroviral vector of the present invention apply equally and without reservation to lentiviral vectors. Furthermore, all references herein to the lentiviral vector of the present invention apply equally and without reservation to retroviral vectors.

[0196] Examples of suitable retroviruses for use in the present invention include gammaretroviruses, such as murine leukemia virus (MLV) and feline leukemia virus (FLV). Examples of suitable lentiviruses for use in the present invention include simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), and visna / maedi virus. Preferably, the present invention relates to lentiviral vectors and their production. Particularly preferred lentiviral vectors are SIV vectors (including all strains and subtypes), such as SIV-AGM (originally isolated from African green monkeys, or savanna monkeys). Alternatively, the present invention relates to HIV vectors.

[0197] Retroviral / lentiviral (e.g., SIV) vectors of the present invention are typically pseudotyped with the hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus or the G glycoprotein from vesicular stomatitis virus (referred to as VSV-G or G-VSV). Preferably, lentiviral (e.g., SIV) vectors of the present invention are pseudotyped with the HN and F proteins from a respiratory paramyxovirus. Particularly preferably, the respiratory paramyxovirus is Sendai virus (murine parainfluenza virus type 1). Retroviral / lentiviral (e.g., SIV) vectors of the present invention may also be pseudotyped with proteins from another virus, provided that the pseudotyping protein does not negatively affect (or even increase) the titer of the produced vector and / or negatively affect (or even increase) transgene expression. Non-limiting examples of other proteins that can be used to pseudotype the retroviral / lentiviral (e.g., SIV) vectors of the invention include the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein or modified forms thereof. VSV-G and SARS-CoV2 spike proteins used for pseudotyping include those described in UK Patent Application No. 2118685.3 and International Application No. PCT / GB2022 / 050933, each of which is incorporated herein by reference in its entirety.

[0198] Retroviral / lentiviral (e.g., SIV) vectors for use according to the present invention may be integrase-competent (IC). Alternatively, lentiviral (e.g., SIV) vectors may be integrase-deficient (ID).

[0199] The viral vectors of the present invention, particularly the retroviral / lentiviral (e.g., SIV) vectors described herein, can be transduced into one or more cell types described herein to achieve repeated transient GM-CSF expression over long periods of time.

[0200] The nucleic acid sequence encoding the therapeutic protein to be included in the retroviral / lentiviral (e.g., SIV) vector of the present invention may be modified to enhance expression.For example, the transgene sequence may be CpG-deleted (or CpG-free) and / or codon-optimized to enhance gene expression.Standard techniques for modifying transgene sequences in this way are known in the art.The genome of the retroviral / lentiviral (e.g., SIV) vector may be completely or partially CpG-deleted (or CpG-free) and / or codon-optimized.

[0201] Retroviral / lentiviral (e.g., SIV) vectors, such as those of the present invention, can be integrated into the genome of transduced cells, resulting in the ability to repeatedly express transiently over a long period of time, making them suitable for transducing stem / progenitor cells.In the lung, several cell types with regenerative capacity have been identified that are responsible for maintaining specific cell lineages in the ductal airways and alveoli.These include basal cells and submucosal gland duct cells in the upper respiratory tract, club cells and neuroendocrine cells in the bronchiolar airways, bronchoalveolar stem cells in the terminal bronchioles, and type II pneumocytes in the alveoli. Thus, without being bound by theory, it is believed that the retroviral / lentiviral (e.g., SIV) vectors allow for long-term transient GM-CSF expression by introducing a transgene into one or more long-lived airway epithelial cells or cell types, such as basal cells and submucosal gland duct cells in the upper respiratory tract, club cells and neuroendocrine cells in the bronchiolar airways, bronchoalveolar stem cells in the terminal bronchioles, type II pneumocytes, submucosal acinar cells, ionocytes, and type I pneumocytes in the alveoli.

[0202] Therefore, the retroviral / lentiviral (e.g., SIV) vector of the present invention can be transduced into one or more cells or cell lines with regenerative capacity in the lungs (including the airways and respiratory tract) to allow long-term transient GM-CSF expression. For example, the retroviral / lentiviral (e.g., SIV) vector can be transduced into basal cells, such as those in the upper airways / respiratory tract. Basal cells play a central role in the process of epithelial maintenance and repair after injury. In addition, basal cells are widely distributed in human respiratory epithelium, with a relative distribution range of 30% (large airways) to 6% (small airways).

[0203] The retroviral / lentiviral (e.g., SIV) vectors of the invention can be used to transduce isolated and expanded stem / progenitor cells ex vivo prior to administration to a patient. Preferably, the retroviral / lentiviral (e.g., SIV) vectors of the invention are used to transduce cells within the lung (or airway / respiratory tract) in vivo.

[0204] The retroviral / lentiviral (e.g., SIV) vectors of the present invention exhibit remarkable resistance to shear forces and have only a slight reduction in transduction capacity when passed through clinically relevant delivery devices, such as bronchoscopes, spray bottles, and nebulizers.

[0205] The retroviral / lentiviral vectors of the present invention enable the expression of therapeutic levels of GM-CSF (particularly free GM-CSF). The retroviral / lentiviral (e.g., SIV) vectors of the present invention typically provide therapeutic levels of GM-CSF expression when administered to a patient. Expression can be measured by any appropriate method (qualitative or quantitative, preferably quantitative), and concentrations are provided in any appropriate units of measurement, such as ng / ml or nM. As described herein, therapeutic levels or concentrations of GM-CSF expression are relatively low, and potentially even below the lower limit of detection when quantifying GM-CSF levels using standard assays, such as bronchoalveolar lavage fluid (BALF) or lung tissue. However, therapeutic effects can still be quantified based on parameters such as BALF turbidity, surfactant protein D (SF-D) concentration in BALF or lungs, surfactant deposition in the lungs, CT scans, and / or pulmonary function metrics, as described herein.

[0206] Preferably, the present invention relates to F / HN retroviral / lentiviral vectors, in particular SIV F / HN vectors, comprising a promoter and a GM-CSF transgene.

[0207] The retroviral / lentiviral (e.g., SIV) vectors of the present invention may be deleted for their endogenous Rev response element (RRE) genomic element, with a retroviral RRE inserted into the intron within 100 bp 5' of the intron's splice acceptor branch site. The intron may be a chimeric intron as described herein, e.g., a β-globin / IgG chimeric intron. Such a β-globin / IgG chimeric intron containing a retroviral / lentiviral RRE is described in UK Patent Application No. 2213936.4 (although not in the context of providing transient and / or low-level GM-CSF expression to provide GM-CSF within the narrow therapeutic window taught for the first time herein). UK Patent Application No. 2213936.4 is incorporated herein by reference in its entirety.

[0208] The viral vector of the present invention can be produced by any suitable process known in the art.Specifically, the retroviral / lentiviral (e.g., SIV) vector of the present invention can be produced by the method disclosed in International Application No. PCT / GB2022 / 050524, which is incorporated herein by reference in its entirety.

[0209] Viral vectors of the present invention, particularly retroviral / lentiviral (e.g., SIV) vectors of the present invention, can include a central polypurine tract (cPPT) and / or a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). An exemplary WPRE sequence is provided by SEQ ID NO: 12.

[0210] The present invention also provides a host cell comprising the retroviral / lentiviral (e.g., SIV) vector of the present invention. Typically, the host cell is a mammalian cell, particularly a human cell or cell line. Non-limiting examples of host cells include HEK293 cells (e.g., HEK293F or HEK293T cells) and 293T / 17 cells. Commercially available cell lines suitable for virus production are also readily available (described herein).

[0211] Inducible promoters Expression of the GM-CSF transgene according to the present invention can be controlled using an inducible promoter (also referred to interchangeably herein as a regulatable promoter).

[0212] Inducible promoters can be used in the non-viral nucleic acids of the invention, particularly the plasmids described herein. Inducible promoters are used in the viral vectors of the invention.

[0213] The use of an inducible promoter allows for the control of GM-CSF expression in target cells. Specifically, the use of an inducible promoter allows for the control of the duration and / or level of GM-CSF expression. Thus, the use of an inducible promoter allows for the duration of GM-CSF expression in a patient's cells and / or the concentration of GM-CSF expressed in a patient's cells to be carefully controlled, allowing for transient and / or low-level expression, resulting in GM-CSF being expressed within the narrow toxicity / efficacy window described herein. Thus, an inducible promoter can facilitate the treatment of PAP described herein, maintaining the concentration of GM-CSF (especially free GM-CSF) within a narrow therapeutic window, achieving a therapeutic effect on the patient while reducing and / or eliminating the histopathological changes in the patient that are typically associated with prolonged and / or high-level GM-CSF expression.

[0214] As used herein, the term "inducible promoter" refers to a promoter that initiates transcription only when stimulated, typically by an exogenous stimulus. An inducible promoter can be regulated by an exogenous factor (also called an inducer), such as a steroid, a chemical inducer of dimerization, or other inducer, and can initiate transcription only when stimulated by the inducer.

[0215] An inducible promoter can be positively inducible, whereby in the off state, the promoter is inactive because its activator protein cannot bind to it. After the inducer binds to the activator protein, the activator protein can bind to the promoter, turning it on and initiating transcription.

[0216] An inducible promoter may be negatively inducible, whereby in the off state, the promoter is inactive because a bound repressor protein actively prevents transcription. When the inducer binds to the repressor protein, the repressor protein is removed from the DNA. In the absence of the repressor protein, transcription is turned on.

[0217] Chemical agents, temperature, and light are all examples of factors that can result in the induction of a promoter. Other non-limiting examples of inducible promoters that can be used in accordance with the present invention are synthetic promoters that rely on endogenous transcription elements (such as those produced by Sympromics-AskBio).

[0218] Non-limiting examples of chemically regulated / chemically inducible promoters include steroid-regulated promoters (e.g., the AlcA promoter activated by AlcR) and alcohol-regulated promoters (e.g., the LexA promoter activated by XVE, or the GeneSwitch™ system by Thermo Fisher). The rapamycin-induced dimerization system is another example of a chemically regulated promoter.

[0219] Non-limiting examples of temperature-inducible / temperature-regulated promoters include promoters from heat shock-inducible Hsp70 or Hsp90, where the selected gene is expressed only after exposure to a mild heat shock.

[0220] Typically, the inducible promoter of the present invention can be regulated by a chemical agent / inducer. Chemically regulated promoters are typically easily induced in an in vivo / therapeutic environment. A regulated promoter system typically includes a regulated promoter (which can replace hCEF or any of the other promoters described herein) and a transactivator (encoded by a regulated plasmid or a plasmid of the present invention). As a non-limiting example, in the context of a retroviral / lentiviral (e.g., SIV) vector of the present invention, the vector genome plasmid (pDNA1) can contain a GM-CSF transgene operably linked to a regulated promoter. pDNA1 may further encode the corresponding transactivator. Thus, the GM-CSF transgene operably linked to the regulated promoter and the transactivator can be encoded by a single retroviral / lentiviral (e.g., SIV) vector. In a single retroviral / lentiviral (e.g., SIV) vector system, (i) the GM-CSF transgene operably linked to the regulated promoter and (ii) the gene encoding the transactivator are present in the same vector backbone, typically in opposite orientations. Alternatively, the GM-CSF transgene operably linked to a regulated promoter is encoded by a first retroviral / lentiviral (e.g., SIV) vector, and the transactivator is encoded by a second retroviral / lentiviral (e.g., SIV) vector. Preferably, a two-vector system is used, i.e., the transactivator is encoded by a retroviral / lentiviral (e.g., SIV) vector separate from the GM-CSF transgene operably linked to the second / regulated promoter. As a further non-limiting example, with respect to the non-viral nucleic acid molecules of the present invention, e.g., plasmids for delivery to a patient, the non-viral nucleic acid molecule (e.g., plasmid) can comprise a GM-CSF transgene operably linked to a regulated promoter. The non-viral nucleic acid molecule (e.g., plasmid) can further encode the corresponding transactivator.Therefore, the GM-CSF transgene operably linked to the regulated promoter and the transactivator are encoded by a single non-viral nucleic acid molecule (e.g., a plasmid).In a single non-viral nucleic acid molecule (e.g., a plasmid) system, (i) the GM-CSF transgene operably linked to the regulated promoter and (ii) the gene encoding the transactivator are present in the same non-viral nucleic acid molecule (e.g., a plasmid), typically in opposite orientations.Alternatively, the GM-CSF transgene operably linked to the regulated promoter is encoded by a first non-viral nucleic acid molecule (e.g., a plasmid), and the transactivator is encoded by a second non-viral nucleic acid molecule (e.g., a plasmid).Any of these non-viral nucleic acid molecules (e.g., a plasmid) can be included in the lipid carrier described herein for delivery to patients.

[0221] In some embodiments, steroid-regulated promoters can be used.Steroid-regulated promoter systems are known in the art, and suitable systems are commercially available (for example, the GeneSwitch™ system by Thermo Fisher).It is within the routine of those skilled in the art to use such steroid-regulated promoters in the non-viral nucleic acid molecules and viral / retroviral / lentiviral (for example, SIV) vectors of the present invention.

[0222] A steroid-regulated promoter system typically includes a steroid-regulated promoter (which can replace hCEF or any of the other promoters described herein) and a transactivator (encoded by a regulatory plasmid or a plasmid of the present invention). As a non-limiting example, in the context of a retroviral / lentiviral (e.g., SIV) vector of the present invention, the vector genome plasmid (pDNA1) can include a GM-CSF transgene operably linked to a steroid-regulated promoter. pDNA1 may further encode the corresponding transactivator. Thus, a GM-CSF transgene operably linked to a steroid-regulated promoter and a transactivator can be encoded by a single retroviral / lentiviral (e.g., SIV) vector. In a single retroviral / lentiviral (e.g., SIV) vector system, (i) a GM-CSF transgene operably linked to a steroid-regulated promoter, and (ii) a gene encoding a transactivator are present in the same vector backbone, typically in opposite orientations. Alternatively, the GM-CSF transgene operably linked to a steroid-regulated promoter is encoded by a first retroviral / lentiviral (e.g., SIV) vector, and the transactivator is encoded by a second retroviral / lentiviral (e.g., SIV) vector. Preferably, a two-vector system is used, i.e., the transactivator is encoded by a separate retroviral / lentiviral (e.g., SIV) vector from the GM-CSF transgene operably linked to a second / steroid-regulated promoter. As a further non-limiting example, with respect to the non-viral nucleic acid molecules of the invention, e.g., plasmids for delivery to a patient, the non-viral nucleic acid molecule (e.g., plasmid) can comprise a GM-CSF transgene operably linked to a steroid-regulated promoter. The non-viral nucleic acid molecule (e.g., plasmid) can further encode a corresponding transactivator.Therefore, the GM-CSF transgene operably linked to the steroid-regulated promoter and the transactivator can be encoded by a single non-viral nucleic acid molecule (e.g., a plasmid).In a single non-viral nucleic acid molecule (e.g., a plasmid), (i) the GM-CSF transgene operably linked to the steroid-regulated promoter and (ii) the gene encoding the transactivator are present in the same non-viral nucleic acid molecule (e.g., a plasmid), typically in opposite orientations.Alternatively, the GM-CSF transgene operably linked to the steroid-regulated promoter is encoded by a first non-viral nucleic acid molecule (e.g., a plasmid), and the transactivator is encoded by a second non-viral nucleic acid molecule (e.g., a plasmid).Any of these non-viral nucleic acid molecules (e.g., a plasmid) can be included in the lipid carrier described herein for delivery to patients.

[0223] Transactivators typically contain or consist of three parts: (i) a DNA-binding domain composed of zinc fingers; (ii) a drug or ligand-binding domain (which binds to an inducer, e.g., mifepristone); and (iii) an activation domain (e.g., p65) required to activate transgene expression. The activator is present within the gene therapy agent and therefore always present in the target cell after delivery of the gene therapy agent. However, it is only activated if the inducer is also delivered. Once the inducer is delivered, the transactivator becomes functional and seeks its specific DNA-binding site, also known as the zinc finger binding sequence. The GM-CSF transgene cassette under the control of an inducible promoter contains or consists of (i) a zinc finger binding sequence; (ii) a GM-CSF cDNA; and (iii) a bovine growth hormone polyadenylation sequence to promote correct processing of the GM-CSF transgene.

[0224] In patients, GM-CSF transgene expression by target cells can be initiated by administration of an appropriate activator, e.g., an appropriate steroid in the case of a steroid-regulated promoter (e.g., mifepristone in the case of a mifepristone-regulated promoter, e.g., GeneSwitch™, or a vector-variant form of one thereof).

[0225] One non-limiting example of a steroid-regulated promoter that can be used in the present invention is a mifepristone-regulated promoter, such as the commercially available GeneSwitch™. This exemplary mifepristone-regulated promoter has the following structure: (i) a GAL4 upstream activation sequence (UAS) that can contain six GAL4 binding sites; (ii) an adenovirus E1b TATA box; and (iii) an intron (e.g., synthetic intron IVS8). A non-limiting example of a mifepristone-regulated promoter sequence is found in SEQ ID NO: 13. An exemplary transactivator for use with a mifepristone-regulated promoter can have the following structure: (i) a GAL4 DNA binding domain (DBD); (ii) a human progesterone receptor ligand binding domain (lPR-LBD) that binds to mifepristone; and (iii) a human NF-κB p65 activation domain (AD). A non-limiting example of a nucleic acid sequence encoding a transactivator for use with a mifepristone-regulated promoter is found in SEQ ID NO: 14. In this exemplary two-vector system, in the presence of mifepristone, the hPR-LBD domain of the GeneSwitch™ regulatory protein undergoes a structural change, enabling activation of the GAL4-E1b promoter, resulting in transgene expression. The transactivator further upregulates its own expression by binding to the Gal4 DNA binding domain upstream of the HSV TK promoter, thus amplifying the induction of expression of the gene of interest. In an exemplary one-vector system, the upstream regulated promoter of GM-CSF is SEQ ID NO: 13, and the transactivator of SEQ ID NO: 14 is also used. However, the promoter sequence driving expression in the one-vector system is a constitutive promoter, such as hCEF.

[0226] Method of production Also described herein are methods for the production of the retroviral / lentiviral (eg, SIV) vectors of the present invention.

[0227] The present inventors have previously demonstrated that the use of a codon-optimized gal-pol gene derived from SIV does not negatively affect the production titer of SIV vectors pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins derived from respiratory paramyxoviruses, and can even result in increased vector titer. This is described in PCT / GB2022 / 050524, which is incorporated herein by reference in its entirety. Furthermore, the present inventors have shown that retroviral vectors containing retroviral / lentiviral RNA sequences containing (i) codon substitutions and (ii) a reduced number of modified retroviral / lentiviral open reading frames (ORFs) do not negatively affect production vector titer, transgene expression, and / or integration of the retroviral / lentiviral RNA sequences into the host / target cell genome, and can even result in increased vector titer, transgene expression, and / or integration of the retroviral / lentiviral RNA sequences. This is described in UK Application No. 2212472.1, which is incorporated herein by reference in its entirety.

[0228] Thus, the present invention provides methods for producing retroviral / lentiviral (e.g., SIV) vectors comprising a GM-CSF transgene operably linked to an inducible promoter, e.g., an inducible promoter described herein.

[0229] The retrovirus / lentivirus (e.g., SIV) is typically pseudotyped with the hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxoviruses or VSV-G, which contain a promoter and a transgene. Preferably, the retrovirus / lentivirus (e.g., SIV) vector is a lentivirus vector, with simian immunodeficiency virus (SIV) vectors being particularly preferred.

[0230] The method of the present invention can be a scalable GMP compatible method.

[0231] The methods of the present invention allow for the production of high-titer retroviral / lentiviral (e.g., SIV) vectors described herein, which exhibit therapeutic levels of GM-CSF transgene expression. Typically, the methods of the present invention produce retroviral / lentiviral (e.g., SIV) vectors described herein that allow for controlled expression of GM-CSF in target cells. Specifically, the retroviral / lentiviral (e.g., SIV) vectors of the present invention allow for control of the duration and / or level of GM-CSF expression. Thus, by including an inducible promoter in the retroviral / lentiviral (e.g., SIV) vector of the present invention, the duration of GM-CSF expression in the patient's cells and / or the concentration of GM-CSF expressed in the patient's cells can be carefully controlled, allowing for transient and / or low-level expression, resulting in GM-CSF being expressed within the narrow toxicity / efficacy window described herein.

[0232] The methods of the present invention typically allow for the production of retroviral / lentiviral (e.g., SIV) vectors containing modified retroviral / lentiviral (e.g., SIV) RNA sequences with high levels of vector integration into the host / target cell genome. Alternatively, or in addition, the methods of the present invention may allow for the production of high-titer purified retroviral / lentiviral (e.g., SIV) vectors containing modified retroviral / lentiviral (e.g., SIV) RNA sequences. These advantageous properties of the vectors and methods of the present invention are described in UK Application No. 2212472.1, which is incorporated herein by reference in its entirety.

[0233] The production of two retroviral / lentiviral (e.g., SIV) vector systems typically utilizes one or more plasmids that provide the elements required for the production of a vector containing a GM-CSF transgene: the retroviral / lentiviral vector genome, Gag-Pol, Rev, F, and HN. Multiple elements may be provided on a single plasmid. Preferably, each element is provided on a separate plasmid, resulting in five plasmids, one each for the vector genome, Gag-Pol, Rev, F, and HN. Alternatively, a single plasmid may provide the Gag-Pol and Rev elements, which may be referred to as the packaging plasmid (pDNA2). The remaining elements (genome, F, and HN) may be provided by separate plasmids (pDNA1, pDNA3a, and pDNA3b, respectively), resulting in four plasmids being used to produce a retroviral / lentiviral (e.g., SIV) vector containing a GM-CSF transgene according to the present invention. In the four-plasmid approach, pDNA1, pDNA3a, and pDNA3b can be as described herein in the context of the five-plasmid approach. In the two-vector system, the transactivator is encoded by an alternate vector genome plasmid (pDNA1*). The remaining elements can be encoded by (i) the same pDNA2a, pDNA2b, pDNA3a, and pDNA3b used to generate the vector containing the GM-CSF transgene (five-plasmid approach); or (ii) the same pDNA2, pDNA3a, and pDNA3b (four-plasmid approach).

[0234] In the one-vector system, the transgene encoding the transactivator is contained in the same vector genome plasmid (pDNA1) as the GM-CSF transgene. ta+) This vector genome plasmid may be used in a four- or five-plasmid approach to produce a retroviral / lentiviral (e.g., SIV) one-vector system according to the invention. Thus, the remaining elements may be encoded by (i) the same pDNA2a, pDNA2b, pDNA3a, and pDNA3b (five-plasmid approach); or (ii) the same pDNA2, pDNA3a, and pDNA3b (four-plasmid approach) as those used to produce the vector containing the GM-CSF transgene in the two-vector system. For retroviral / lentiviral (e.g., SIV) vectors pseudotyped with an alternative envelope protein, e.g., VSV-G, rather than the F and HN proteins, the method for producing a two-vector system of the invention also typically utilizes one or more plasmids providing the elements required for the production of a vector containing a GM-CSF transgene: the retroviral / lentiviral vector genome, Gag-Pol (pDNA2a), Rev (pDNA2b), and envelope (e.g., VSV-G) (pDNA3). Multiple elements may be provided on a single plasmid. Preferably, each element is provided on a separate plasmid, resulting in four plasmids: one each for the vector genome, Gag-Pol, Rev, and envelope (e.g., VSV-G). In a four-plasmid method for producing a VSV-G-pseudotyped retroviral / lentiviral vector containing a two-vector system GM-CSF transgene, pDNA1, pDNA2a, and pDNA2b may be as described herein in the context of the five-plasmid method for producing two retroviral / lentiviral vector systems pseudotyped with F and HN proteins. Alternatively, a single plasmid may provide the Gag-Pol and Rev elements, which may be referred to as the packaging plasmid (pDNA2).The remaining elements (genome and VSV-G) may be provided by separate plasmids (pDNA1 and pDNA3, respectively), and three plasmids would be used to produce a retroviral / lentiviral (e.g., SIV) vector containing a GM-CSF transgene in a two-vector system according to the present invention. In the three-plasmid approach, pDNA1 can be as described herein in the context of the five / four-plasmid approach. In the two-vector system, the transactivator is encoded by an alternate vector genome plasmid (pDNA1*). The remaining elements may be encoded by (i) the same pDNA2a, pDNA2b, and pDNA3 (four-plasmid approach); or (ii) the same pDNA2 and pDNA3 (three-plasmid approach) as those used to produce the vector containing the GM-CSF transgene.

[0235] In the one-vector system, the transgene encoding the transactivator is contained in the same vector genome plasmid (pDNA1) as the GM-CSF transgene. ta+ ) This vector genome plasmid may be used in a four- or three-plasmid approach to generate a retroviral / lentiviral (e.g., SIV)1 vector system according to the invention. Thus, the remaining elements may be encoded by (i) the same pDNA2a, pDNA2b, and pDNA3 (four-plasmid approach); or (ii) the same pDNA2 and pDNA3 (three-plasmid approach) as those used to generate the vector containing the GM-CSF transgene in a two-vector system.

[0236] Preferably, the vector genome plasmid encodes all genetic material to be packaged into the final retroviral / lentiviral vector, including the transgene. The vector genome plasmid may be referred to herein as "pDNA1" and typically contains the GM-CSF transgene. In a two-vector system, the transactivator is encoded by an alternate vector genome plasmid (pDNA1*). In a one-vector system, the transgene encoding the transactivator is contained in the same vector genome plasmid (pDNA1*) as the GM-CSF transgene. ta+ In a preferred five-plasmid method for producing retroviral / lentiviral vectors containing the GM-CSF transgene, the other plasmids are production plasmids encoding the Gag-Pol, Rev, F, and HN proteins. These plasmids can be designated "pDNA2a," "pDNA2b," "pDNA3a," and "pDNA3b," respectively.

[0237] Typically, the lentivirus is an SIV, such as SIV1, preferably SIV-AGM. The F and HN proteins are derived from a respiratory paramyxovirus, preferably a Sendai virus.

[0238] In a specific embodiment, the five plasmids for producing an SIV vector containing a GM-CSF transgene as part of a two-vector system are characterized by Figures 2B and 2D-H, whereby pDNA1 is the pSIV-2V-GMCSF plasmid of Figure 2B, pDNA2a is the pGM691 plasmid of Figure 2D or the pGM297 plasmid of Figure 2E, pDNA2b is the pGM299 plasmid of Figure 2F, pDNA3a is the pGM301 plasmid of Figure 2G, and pDNA3b is the pGM303 plasmid of Figure 2H, or a variant of any of these plasmids (as described herein).

[0239] The plasmid defined in Figure 2B is represented by SEQ ID NO: 15; the plasmid defined in Figure 2D is represented by SEQ ID NO: 16; the plasmid defined in Figure 2E is represented by SEQ ID NO: 17; the plasmid defined in Figure 2F is represented by SEQ ID NO: 18; the plasmid defined in Figure 2G is represented by SEQ ID NO: 19; and the plasmid defined in Figure 2H is represented by SEQ ID NO: 20. Variants of these plasmids (as defined herein) are also encompassed by the present invention. Specifically, variants having at least 90% (e.g., at least 90, 92, 94, 95, 96, 97, 98, 99, 99.5, or 100%) sequence identity to any one of SEQ ID NOs: 15-20 are encompassed.

[0240] In certain embodiments, the five plasmids for producing a transactivator-containing SIV vector as part of a two-vector system are characterized by Figures 2C-2H, where pDNA1* is the pSIV-2V-transactivator plasmid of Figure 2C, pDNA2a is the pGM691 plasmid of Figure 2D or the pGM297 plasmid of Figure 2E, pDNA2b is the pGM299 plasmid of Figure 2F, pDNA3a is the pGM301 plasmid of Figure 2G, and pDNA3b is the pGM303 plasmid of Figure 2H, or a variant of any of these plasmids (as described herein).

[0241] The plasmid defined in Figure 2C is represented by SEQ ID NO:21; the plasmid defined in Figure 2D is represented by SEQ ID NO:16; the plasmid defined in Figure 2E is represented by SEQ ID NO:17; the plasmid defined in Figure 2F is represented by SEQ ID NO:18; the plasmid defined in Figure 2G is represented by SEQ ID NO:19; and the plasmid defined in Figure 2H is represented by SEQ ID NO:20. Variants of these plasmids (as defined herein) are also encompassed by the present invention. Specifically, variants having at least 90% (e.g., at least 90, 92, 94, 95, 96, 97, 98, 99, 99.5, or 100%) sequence identity to any one of SEQ ID NOs:16-21 are encompassed.

[0242] In a specific embodiment, the five plasmids for producing an SIV vector containing a GM-CSF transgene and a transactivator as part of a one-vector system are characterized by Figures 2A and 2D-2H and are therefore designated pDNA1. ta+ is the pSIV-1V-GM-CSF plasmid of Figure 2A, pDNA2a is the pGM691 plasmid of Figure 2D or the pGM297 plasmid of Figure 2E, pDNA2b is the pGM299 plasmid of Figure 2F, pDNA3a is the pGM301 plasmid of Figure 2G, and pDNA3b is the pGM303 plasmid of Figure 2H, or a variant of any of these plasmids (as described herein).

[0243] The plasmid defined in Figure 2A is represented by SEQ ID NO:22; the plasmid defined in Figure 2D is represented by SEQ ID NO:16; the plasmid defined in Figure 2E is represented by SEQ ID NO:17; the plasmid defined in Figure 2F is represented by SEQ ID NO:18; the plasmid defined in Figure 2G is represented by SEQ ID NO:19; and the plasmid defined in Figure 2H is represented by SEQ ID NO:20. Variants of these plasmids (as defined herein) are also encompassed by the present invention. Specifically, variants having at least 90% (e.g., at least 90, 92, 94, 95, 96, 97, 98, 99, 99.5, or 100%) sequence identity to any one of SEQ ID NOs:16-20 and 22 are encompassed.

[0244] In each of the three-, four-, or five-plasmid methods of the invention, all of the plasmids contribute to the formation of the final retroviral / lentiviral (e.g., SIV) vector (whether encoding the GM-CSF transgene or transactivator in a two-vector system, or both the GM-CSF transgene and transactivator in a one-vector system), but only the vector genome plasmid provides the nucleic acid sequences included in the retroviral / lentiviral (e.g., SIV) RNA sequence. During the manufacture of the retroviral / lentiviral (e.g., SIV) vector, the vector genome plasmid (pDNA1 / pDNA1* / pDNA ta+) provides an enhancer / promoter, Psi, an intron containing RRE, cPPT, mWPRE, SIN LTR, and SV40 polyA (see Figures 2A-C), which are important for virus production. Using pSIV-2V-GMCSF as a non-limiting example of pDNA1, the CMV enhancer / promoter, SV40 polyA, colE1 Ori, and KanR are involved in the production of the retroviral / lentiviral (e.g., SIV) vector of the present invention, but are not found in the final retroviral / lentiviral (e.g., SIV) vector. The cPPT (central polypurine tract), RRE-containing intron (inserted between the hCEF and AAT transgenes), hCEF, AAT (transgene), and mWPRE from pSIV-2V-GMCSF are found in the final retroviral / lentiviral (e.g., SIV) vector. The SIN LTR (long terminal repeat, SIN / IN self-inactivating) and Psi (packaging signal) are found in the final retroviral / lentiviral (eg SIV) vector.

[0245] For other retroviral / lentiviral (e.g., SIV) vectors of the invention, corresponding elements from other vector genome plasmids (pDNA1) are required for production (but not found in the final vector) or are present in the final retroviral / lentiviral (e.g., SIV) vector.

[0246] In a specific embodiment for pseudotyping with VSV-G, four plasmids for producing an SIV vector containing a GM-CSF transgene as part of a two-vector system are characterized by Figures 2B, 2D-2F, and 2I, where pDNA1 is the pSIV-2V-GMCSF plasmid of Figure 2B, pDNA2a is the pGM691 plasmid of Figure 2D or the pGM297 plasmid of Figure 2E, pDNA2b is the pGM299 plasmid of Figure 2F, and pDNA3 is the pMD2.G plasmid of Figure 2I, or a variant of any of these plasmids (as described herein). The plasmid defined in Figure 2B is represented by SEQ ID NO: 15; the plasmid defined in Figure 2D is represented by SEQ ID NO: 16; the plasmid defined in Figure 2E is represented by SEQ ID NO: 17; the plasmid defined in Figure 2F is represented by SEQ ID NO: 18; and the plasmid defined in Figure 2I is represented by SEQ ID NO: 23. Variants of these plasmids (as defined herein) are also encompassed by the present invention. Specifically, variants having at least 90% (eg, at least 90, 92, 94, 95, 96, 97, 98, 99, 99.5, or 100%) sequence identity to any one of SEQ ID NOs: 15-18 and 23 are encompassed.

[0247] In a specific embodiment, the four plasmids for producing a transactivator-containing SIV vector as part of a two-vector system are characterized by Figures 2C-2F and 2I, where pDNA1* is the pSIV-2V-transactivator plasmid of Figure 2C, pDNA2a is the pGM691 plasmid of Figure 2D or the pGM297 plasmid of Figure 2E, pDNA2b is the pGM299 plasmid of Figure 2F, and pDNA3 is the pMD2.G plasmid of Figure 2I, or a variant of any of these plasmids (as described herein).

[0248] The plasmid defined in Figure 2C is represented by SEQ ID NO:21; the plasmid defined in Figure 2D is represented by SEQ ID NO:16; the plasmid defined in Figure 2E is represented by SEQ ID NO:17; the plasmid defined in Figure 2F is represented by SEQ ID NO:18; and the plasmid defined in Figure 2I is represented by SEQ ID NO:23. Variants of these plasmids (as defined herein) are also encompassed by the present invention. Specifically, variants having at least 90% (e.g., at least 90, 92, 94, 95, 96, 97, 98, 99, 99.5, or 100%) sequence identity to any one of SEQ ID NOs:16-18, 21, and 23 are encompassed.

[0249] In a specific embodiment, the four plasmids for producing an SIV vector containing a GM-CSF transgene and a transactivator as part of a one-vector system are characterized by Figures 2A, 2D-2F, and 2I, and are therefore designated pDNA1. ta+ is the pSIV-1V-GM-CSF plasmid of Figure 2A, pDNA2a is the pGM691 plasmid of Figure 2D or the pGM297 plasmid of Figure 2E, pDNA2b is the pGM299 plasmid of Figure 2F, and pDNA3 is the pMD2.G plasmid of Figure 2I, or a variant of any of these plasmids (as described herein).

[0250] The plasmid defined in Figure 2A is represented by SEQ ID NO:22; the plasmid defined in Figure 2D is represented by SEQ ID NO:16; the plasmid defined in Figure 2E is represented by SEQ ID NO:17; the plasmid defined in Figure 2F is represented by SEQ ID NO:18; and the plasmid defined in Figure 2I is represented by SEQ ID NO:23. Variants of these plasmids (as defined herein) are also encompassed by the present invention. Specifically, variants having at least 90% (e.g., at least 90, 92, 94, 95, 96, 97, 98, 99, 99.5, or 100%) sequence identity to any one of SEQ ID NOs:16-18, 22, and 23 are encompassed.

[0251] The F and HN proteins (preferably Sendai F and HN proteins) derived from pDNA3a and pDNA3b, or the VSV-G derived from pDNA3, are important for the final retroviral / lentiviral (e.g., SIV) vector to infect target cells, i.e., for entry into the patient's epithelial cells (typically lung or nasal cells as described herein). The products of the pDNA2a and pDNA2b plasmids (or pDNA2, when the Gag-Pol and Rev elements are combined in a single plasmid) are important for viral transduction, i.e., insertion of retroviral / lentiviral (e.g., SIV) DNA into the host genome. The promoter, regulatory elements (e.g., WPRE), and transgene are important for expression of the transgene in target cells.

[0252] The methods of the present invention may comprise or consist of the following steps: (a) growing cells in suspension; (b) transfecting the cells with one or more plasmids; (c) adding a nuclease; (d) recovering the retrovirus / lentivirus (e.g., SIV); (e) adding trypsin; and (f) purifying the retrovirus / lentivirus (e.g., SIV).

[0253] This method may use the three-, four-, or five-plasmid system described herein. Thus, for the five-plasmid method for producing a vector containing a GM-CSF transgene as part of a two-vector system, one or more of the plasmids may comprise or consist of the vector genome plasmid pDNA1; a Gag-Pol plasmid (e.g., a codon-optimized Gag-Pol plasmid), pDNA2a; a Rev plasmid, pDNA2b; a fusion (F) protein plasmid, pDNA3a; and a hemagglutinin-neuraminidase (HN) plasmid, pDNA3b. pDNA1 may be pSIV-2V-GMCSF. 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 pSIV-2V-GMCSF; pDNA2a is pGM691; pDNA2b is pGM299; pDNA3a is pGM301; and pDNA3b is pGM303.

[0254] Regarding the five-plasmid method for producing a vector containing a transactivator as part of a two-vector system, one or more of the plasmids can comprise or consist of the vector genome plasmid pDNA1*; a Gag-Pol plasmid (e.g., a codon-optimized Gag-Pol plasmid), pDNA2a; a Rev plasmid, pDNA2b; a fusion (F) protein plasmid, pDNA3a; and a hemagglutinin-neuraminidase (HN) plasmid, pDNA3b. pDNA1* can be pSIV-2V-transactivator. pDNA2a can be pGM297 or pGM691, preferably pGM691. pDNA2b can be pGM299. pDNA3a can be pGM301. pDNA3b can be pGM303. Any combination of pDNA1*, pDNA2a, pDNA2b, pDNA3a, and pDNA3b can be used. Preferably, pDNA1* is pSIV-2V-transactivator; pDNA2a is pGM691; pDNA2b is pGM299; pDNA3a is pGM301; and pDNA3b is pGM303.

[0255] For the five-plasmid method for producing a vector containing a transactivator as part of a one-vector system, one or more plasmids may be the vector genome plasmid pDNA1. ta+ pDNA1 may comprise or consist of a Gag-Pol plasmid (e.g., a codon-optimized Gag-Pol plasmid), pDNA2a; a Rev plasmid, pDNA2b; a fusion (F) protein plasmid, pDNA3a; and a hemagglutinin-neuraminidase (HN) plasmid, pDNA3b. ta+ pDNA2a may be pGM297 or pGM691, preferably pGM691. pDNA2b may be pGM299. pDNA3a may be pGM301. pDNA3b may be pGM303. pDNA1 ta+Any combination of pDNA1, pDNA2a, pDNA2b, pDNA3a, and pDNA3b may be used. ta+ is pSIV-1V-GMCSF; pDNA2a is pGM691; pDNA2b is pGM299; pDNA3a is pGM301; and pDNA3b is pGM303.

[0256] Regarding the four-plasmid method for producing a vector containing a GM-CSF transgene as part of a two-vector system, one or more of the plasmids can comprise or consist of the vector genome plasmid pDNA1; a Gag-Pol plasmid (e.g., a codon-optimized Gag-Pol plasmid), pDNA2a; a Rev plasmid, pDNA2b; and a VSV-G plasmid, pDNA3. pDNA1 can be pSIV-2V-GMCSF. pDNA2a can be pGM297 or pGM691, preferably pGM691. pDNA2b can be pGM299. pDNA3 can be pMD2.G. Any combination of pDNA1, pDNA2a, pDNA2b, and pDNA3 can be used. Preferably, pDNA1 is pSIV-2V-GMCSF; pDNA2a is pGM691; pDNA2b is pGM299; pDNA3a is pGM301; and pDNA3 is pMD2.G.

[0257] For the four-plasmid method for producing a vector containing a transactivator as part of a two-vector system, one or more plasmids can comprise or consist of the vector genome plasmid pDNA1*; a Gag-Pol plasmid (e.g., a codon-optimized Gag-Pol plasmid), pDNA2a; a Rev plasmid, pDNA2b; and a VSV-G plasmid, pDNA3. pDNA1* can be pSIV-2V-transactivator. pDNA2a can be pGM297 or pGM691, preferably pGM691. pDNA2b can be pGM299. pDNA3 can be pMD2.G. Any combination of pDNA1*, pDNA2a, pDNA2b, and pDNA3 can be used. Preferably, pDNA1* is pSIV-2V-transactivator; pDNA2a is pGM691; pDNA2b is pGM299; and pDNA3 is pMD2.G.

[0258] For the four-plasmid method for producing a vector containing a transactivator as part of a one-vector system, one or more of the plasmids may be the vector genome plasmid pDNA1. ta+ pDNA1 may comprise or consist of a Gag-Pol plasmid (e.g., a codon-optimized Gag-Pol plasmid), pDNA2a; a Rev plasmid, pDNA2b; and a VSV-G plasmid, pDNA3. ta+ pDNA2a can be pGM297 or pGM691, preferably pGM691. pDNA2b can be pGM299. pDNA3 can be pMD2.G. pDNA1 ta+ Any combination of pDNA1, pDNA2a, pDNA2b, and pDNA3 may be used. ta+ is pSIV-1V-GMCSF; pDNA2a is pGM691; pDNA2b is pGM299; and pDNA3 is pMD2.G.

[0259] As described herein, a regulated promoter system, e.g., a steroid-regulated promoter system, typically comprises a regulated promoter and a transactivator. Preferably, the vector genome plasmid (pDNA1* for a two-vector system or pDNA1* for a two-vector system) ta+ ) contains a GM-CSF transgene operably linked to a regulatable promoter, as exemplified in pSIV-2V-GMCSF (FIG. 2B and SEQ ID NO: 15) and pSIV-1V-GMCSF (FIG. 2A and SEQ ID NO: 23). pDNA1, i.e., pDNA1 ta+ pSIV-1V-GMCSF may further encode a corresponding transactivator, as exemplified in pSIV-1V-GMCSF (FIG. 2A and SEQ ID NO: 23). Thus, a transgene operably linked to a regulatable promoter and a transactivator are encoded by a single lentiviral (e.g., SIV) vector, which can be produced by the methods of the present invention. In a single vector system, (i) a GM-CSF transgene operably linked to a regulatable promoter, and (ii) a gene encoding the transactivator are present in the same vector backbone, typically in opposite orientations. Alternatively, a transgene operably linked to a regulatable promoter is encoded by a first lentiviral (e.g., SIV) vector, and a transactivator is encoded by a second lentiviral (e.g., SIV) vector.

[0260] Any appropriate ratio of vector genome plasmid:Gag-Pol plasmid:Rev plasmid:F plasmid:HN plasmid can be used to further optimize (increase) the titer of the resulting retrovirus / lentivirus (e.g., SIV). As a non-limiting example, the ratio of vector genome plasmid:Gag-Pol plasmid:Rev plasmid:F plasmid:HN plasmid can be in the range of 10:40:-4 to 20:3 to 12:3 to 12:3 to 12, typically 15:20:7 to 11:4 to 8:4 to 8:4 to 8, e.g., approximately 18:22:7 to 11:4 to 8:4 to 8:4 to 8, or 19:21:8 to 10:5 to 7:5 to 7:5 to 7. Preferably, the ratio of vector genome plasmid:Gag-Pol plasmid:Rev plasmid:F plasmid:HN plasmid is approximately 20:9:6:6:6. Preferably, the ratio of vector genome plasmid:Gag-Pol plasmid:Rev plasmid:VSV-G plasmid is about 20:9:6:12. Steps (a) through (f) of this method are typically performed sequentially, starting with step (a) and continuing through step (f). This method may include one or more additional steps, such as an additional purification step, buffer exchange, concentration of the purified retroviral / lentiviral (e.g., SIV) vector, and / or formulation of the purified (or concentrated) retroviral / lentiviral (e.g., SIV) vector. Each step may include one or more substeps. For example, recovery may include one or more steps or substeps, and / or purification may include one or more steps or substeps.

[0261] Any suitable cell type can be transfected with one or more plasmids (e.g., five, four, or three plasmids described herein) to produce the retroviral / lentiviral (e.g., SIV) vectors of the present invention. Mammalian cells, particularly human cell lines, are typically used. Non-limiting examples of cells suitable for use in the methods of the present invention are HEK293 cells (e.g., HEK293F or HEK293T cells) and 293T / 17 cells. Commercially available cell lines suitable for virus production are also readily available (e.g., Gibco Viral Production Cells—catalog number A35347, from ThermoFisher Scientific). Cells can be grown in animal component-free media, including serum-free media. Cells may also be grown in media containing human components. Cells may also be grown in defined media containing or consisting of synthetically produced components.

[0262] Any suitable transfection means can be used in accordance with the present invention. The selection of a suitable transfection means is within the routine of one skilled in the art. As a non-limiting example, transfection can be performed using PEIPro™, Lipofectamine 2000™, or Lipofectamine 3000™.

[0263] Any suitable nuclease can be used according to the present invention. The selection of a suitable nuclease is within the routine of a person 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 occur before or after recovery, or during the recovery process.

[0264] The gag-pol gene used in producing the retroviral / lentiviral (e.g., SIV) vectors of the present invention may be codon-optimized. Thus, the gag-pol gene in the pDNA2a plasmid is 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:24, or a variant thereof (as defined herein). Specifically, the codon-optimized gag-pol gene of the present invention may comprise or consist of a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO:24, preferably at least 95% identity to SEQ ID NO:24. The codon-optimized gag-pol gene may consist of the nucleic acid sequence of SEQ ID NO:24. A preferred pDNA2a, pGM691, contains the codon-optimized gag-pol gene of SEQ ID NO:24.

[0265] Gag-pol genes (e.g., SIV gag-pol genes), including codon-optimized gag-pol genes, are typically operably linked to a promoter to drive expression of the gag-pol protein. Any suitable promoter can be used, including those described herein in the context of transgene promoters. Preferably, the promoter is the CAG promoter used in the exemplified pGM691 plasmid. An exemplary CAG promoter is set forth in SEQ ID NO: 25. The codon-optimized gag-pol gene of SEQ ID NO: 24 contains translation slippage and therefore does not form a single conventional open reading frame.

[0266] A codon-optimized gag-pol gene (or a nucleic acid comprising or consisting of the same) and a plasmid comprising the gene or nucleic acid are useful in producing retroviral / lentiviral (e.g., SIV) vectors using the methods of the present invention, since they enable the production of high-titer retroviral / lentiviral (e.g., SIV) vectors. Typically, the codon-optimized gag-pol gene (or a nucleic acid comprising or consisting of the same) and a plasmid comprising the gene or nucleic acid can be used to produce a retroviral / lentiviral (e.g., SIV) vector with a titer at least equivalent to that of a retroviral / lentiviral (e.g., SIV) vector produced by a corresponding method that does not use a codon-optimized gag-pol gene, as described herein. The codon-optimized gag-pol gene is further disclosed in PCT / GB2022 / 050524, which is incorporated herein by reference in its entirety.

[0267] The present invention also provides retroviral / lentiviral (eg, SIV) vectors obtainable by the methods of the present invention.

[0268] Typically, retroviral / lentiviral (e.g., SIV) vectors obtainable by the methods of the present invention are produced at high titers, as described herein, which can be measured in transducing units, as defined herein.

[0269] Thus, retroviral / lentiviral (e.g., SIV) vectors of the invention, including those obtainable by the methods of the invention, optionally contain at least about 2.5 x 10 6 TU / mL, at least approximately 3.0 × 10 6 TU / mL, at least approximately 3.1 × 10 6 TU / mL, at least approximately 3.2 × 10 6 TU / mL, at least approximately 3.3 × 10 6 TU / mL, at least approximately 3.4 × 10 6TU / mL, at least approximately 3.5 × 10 6 TU / mL, at least approximately 3.6 × 10 6 TU / mL, at least approximately 3.7 × 10 6 TU / mL, at least approximately 3.8 × 10 6 TU / mL, at least approximately 3.9 × 10 6 TU / mL, at least approximately 4.0 × 10 6 Preferably, the retroviral / lentiviral (e.g., SIV) vector has a titer of at least about 3.0 x 10 TU / mL. 6 TU / mL or at least about 3.5 × 10 6 It is produced at a titer of TU / mL.

[0270] Producing high-titer retroviral / lentiviral (e.g., SIV) vectors can impart other desirable properties to the resulting vector product. For example, without being bound by theory, production at high titers without the need for intensive concentration by methods such as TFF is believed to result in higher quality vector product than retroviral / lentiviral (e.g., SIV) vectors produced by corresponding methods that do not use codon-optimized gag-pol genes (and optionally modified vector genome plasmids) because the vectors are exposed to less shear forces that can damage viral particles and their RNA cargo.

[0271] Typically, the gag-pol gene (e.g., a codon-optimized gag-pol gene) used matches the retroviral / lentiviral vector being produced. As a non-limiting example, if the lentiviral vector is an HIV vector, the codon-optimized gag-pol gene used is the HIV gag-pol gene. As a non-limiting example, if the lentiviral vector is an SIV vector, the codon-optimized gag-pol gene used is the SIV gag-pol gene.

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

[0273] As used herein, the term "trypsin" refers to both trypsin and its equivalents. Equivalent enzymes have the same or essentially the same cleavage specificity as trypsin. Trypsin cleavage activity is defined as cleavage on the C-terminal side of arginine or lysine residues, typically exclusively on the C-terminal side of arginine or lysine residues. Trypsin activity can preferably be provided by a recombinant enzyme free of animal origin, such as TrypLE Select™. Trypsin addition can occur before or after harvesting, or during the harvesting process.

[0274] Retroviral / lentiviral (e.g., SIV) vectors can be purified using appropriate purification methods. Non-limiting examples of suitable purification steps include depth / terminal filtration, tangential flow filtration (TFF), and chromatography. The purification step typically includes at least one chromatography step. Non-limiting examples of chromatography steps that can be used according to the present invention include mixed-mode size exclusion chromatography (SEC) and / or anion exchange chromatography. Elution can be performed with or without, preferably without, the use of a salt gradient.

[0275] Treatment indications The viral / retroviral / lentiviral (e.g., SIV) vectors and non-viral nucleic acid molecules (e.g., plasmids, mRNA, or saRNA) of the present invention can effectively treat PAP by providing a GM-CSF transgene for disease correction.Therefore, the viral / retroviral / lentiviral (e.g., SIV) vectors and non-viral nucleic acid molecules (e.g., plasmids, mRNA, or saRNA) of the present invention can be used to treat PAP (particularly aPAP) by gene therapy, typically with a GM-CSF transgene as described herein.Therefore, the viral / retroviral / lentiviral (e.g., SIV) vectors and non-viral nucleic acid molecules (e.g., plasmids, mRNA, or saRNA) of the present invention can be considered as gene therapy agents, particularly GM-CSF gene therapy agents, or can be included in gene therapy agents, particularly GM-CSF gene therapy agents.

[0276] Therefore, the present invention provides a method for treating PAP, particularly aPAP, comprising administering the viral / retroviral / lentiviral (e.g., SIV) vector and / or non-viral nucleic acid molecule (e.g., plasmid, mRNA, or saRNA) of the present invention to a patient in need thereof.The viral / retroviral / lentiviral (e.g., SIV) vector and / or non-viral nucleic acid molecule (e.g., plasmid, mRNA, or saRNA) of the present invention is typically administered to a patient in a therapeutically effective amount, and the therapeutically effective amount can be easily determined by a clinician without undue burden.Typically, the viral / retroviral / lentiviral (e.g., SIV) vector and / or non-viral nucleic acid molecule (e.g., plasmid, mRNA, or saRNA) of the present invention is produced using the method of the present invention.

[0277] The present invention also provides the viral / retroviral / lentiviral (e.g., SIV) vectors and / or non-viral nucleic acid molecules (e.g., plasmids, mRNA, or saRNA) of the present invention for use in methods for treating diseases, particularly PAP, preferably aPAP. Typically, the viral / retroviral / lentiviral (e.g., SIV) vectors and / or non-viral nucleic acid molecules (e.g., plasmids, mRNA, or saRNA) of the present invention are produced using the methods of the present disclosure.

[0278] The present invention also provides the use of the viral / retroviral / lentiviral (e.g., SIV) vectors and / or non-viral nucleic acid molecules (e.g., plasmids, mRNA, or saRNA) of the present invention in the manufacture of a medicament for use in a method for treating a disease, particularly PAP, preferably aPAP. Typically, the viral / retroviral / lentiviral (e.g., SIV) vectors and / or non-viral nucleic acid molecules (e.g., plasmids, mRNA, or saRNA) of the present invention are produced using the methods of the present disclosure.

[0279] Formulations, Compositions, and Administration The viral / retroviral / lentiviral (e.g., SIV) vectors and / or non-viral nucleic acid molecules (e.g., plasmids, mRNA, or saRNA) of the present invention can be administered at any dosage appropriate to achieve the desired therapeutic effect. As described herein, the dose of the viral / retroviral / lentiviral (e.g., SIV) vectors and / or non-viral nucleic acid molecules (e.g., plasmids, mRNA, or saRNA) of the present invention administered is typically lower than that used for other transgenes (e.g., CFTR or AAT) due to the narrow therapeutic window of GM-CSF. Appropriate dosages are determined by clinicians or other medical professionals using standard techniques and are within their normal scope of practice. Thus, a non-limiting example of a suitable dosage of a viral / retroviral / lentiviral (e.g., SIV) vector is 1 x 10 7Transducing units (TU), 1 × 10 8 TU, 1×10 9 TU, e.g., 1 x 10 7 TU~1×10 8 Any dose within the range of TU is included.

[0280] The present invention also provides compositions comprising the viral / retroviral / lentiviral (e.g., SIV) vectors and / or non-viral nucleic acid molecules (e.g., plasmids, mRNA, or saRNA) of the present invention and a pharmaceutically acceptable carrier. Non-limiting examples of pharmaceutically acceptable carriers include water, saline, and phosphate-buffered saline. In some embodiments, however, the composition is in a lyophilized form, in which case it may contain a stabilizer, such as bovine serum albumin (BSA). In some embodiments, it may be desirable to formulate the composition with a preservative, such as thiomersal or sodium azide, to facilitate long-term storage.

[0281] The viral / retroviral / lentiviral (e.g., SIV) vector and / or non-viral nucleic acid molecule (e.g., plasmid, mRNA, or saRNA) of the present invention can be administered by any suitable route. It may be desirable to direct the composition of the present invention to the subject's respiratory system (as described above). Efficient delivery of the therapeutic / prophylactic composition or medicament to the respiratory infection site can be achieved by oral or intranasal administration, for example, aerosol (e.g., nasal spray), or catheter. Typically, the viral / retroviral / lentiviral (e.g., SIV) vector and / or non-viral nucleic acid molecule (e.g., plasmid, mRNA, or saRNA) of the present invention is stable in clinically relevant nebulizers, inhalers (including metered-dose inhalers), catheters, aerosols, etc. Typically, therefore, the viral / retroviral / lentiviral (e.g., SIV) vectors and / or non-viral nucleic acid molecules (e.g., plasmids, mRNA, or saRNA) of the present invention are formulated for administration to the lungs by any suitable means, for example, they can be formulated for intratracheal administration (e.g., intratracheal instillation), intranasal administration (e.g., intranasal instillation), aerosol delivery, nebulization, or direct injection or delivery to the lungs (e.g., delivered by catheter).Other delivery modes, such as intravenous delivery, are also encompassed by the present invention.

[0282] In some embodiments, the nose is a preferred site for producing therapeutic proteins using viral / retroviral / lentiviral (e.g., SIV) vectors and / or non-viral nucleic acid molecules (e.g., plasmids, mRNA, or saRNA) of the present invention due to at least one of the following reasons: (i) extracellular barriers, such as inflammatory cells and sputum, are less prominent in the nose; (ii) vector administration is easier; (iii) the amount of vector / nucleic acid required is smaller; and (iv) ethical considerations. Thus, transduction of nasal epithelial cells with viral / retroviral / lentiviral (e.g., SIV) vectors or transfection of non-viral nucleic acid molecules (e.g., plasmids, mRNA, or saRNA) of the present invention can result in efficient expression of therapeutic GM-CSF transgenes, as described herein. Therefore, intranasal administration of viral / retroviral / lentiviral (e.g., SIV) vectors and / or non-viral nucleic acid molecules (e.g., plasmids, mRNA, or saRNA) of the present invention may be preferred.

[0283] Formulations for intranasal administration can be in the form of nasal drops or nasal sprays. Nasal formulations can include droplets with approximate diameters ranging from 1 to 5,000 μm, e.g., 500 to 4,000 μm, 1,000 to 3,000 μm, 100 to 1,000 μm, less than 500 μm, less than 400 μm, less than 300 μm, less than 250 μm, less than 200 μm, less than 100 μm, less than 75 μm, less than 50 μm, less than 25 μm, less than 20 μm, less than 15 μm, less than 12.5 μm, less than 10 μm, less than 5 μm, less than 2.5 μm, or less. Alternatively, the volume of the droplets can be within the range of about 0.001 to 100 μl, e.g., 0.1 to 50 μl or 1.0 to 25 μl, or e.g., 0.001 to 1 μl.

[0284] Aerosolized pharmaceutical compositions of the invention can be characterized as having a mass median aerodynamic diameter (MMAD) of less than 5 μm and a fine particle fraction (FPF, defined as the fraction of aerosol contained within droplets having an MMAD of less than 5 μm) of greater than 50%; and having droplet sizes such that greater than 50% of the total aerosolized plasmid is delivered intact.

[0285] Mass median aerodynamic diameter (MMAD) is a well-known means of characterizing droplet size in aerosols. This measurement, along with the geometric standard deviation, is used to statistically describe the droplet size distribution of any aerosol based on droplet weight and size. Means for calculating the MMAD of an aerosol are well known in the art.

[0286] The fine particle fraction is a measure of the proportion of droplets having the desired size characteristics. For the present invention, it is defined as the proportion of the aerosol contained in droplets with a diameter of 1 to 3 μm. Again, means for calculating the fine particle fraction of an aerosol are well known in the art.

[0287] Preferably, the aerosolized pharmaceutical composition is formulated as an aerosol, wherein the aerosol has an MMAD in the range of 1-3 μm and an FPF of greater than 50%; and has a droplet size such that greater than 50% of the total aerosolized droplets are delivered intact. More preferably, the aerosol has an MMAD in the range of 1-3 μm and an FPF of greater than 60%.

[0288] Aerosol formulations can take the form of a powder, suspension, or solution. The size of the aerosol droplets is related to the delivery capacity of the aerosol. Smaller droplets can travel farther from the respiratory tract toward the alveoli than larger droplets. In one embodiment, the aerosol droplets have a diameter distribution that promotes delivery along the entire length of the bronchi, bronchioles, and alveoli. Alternatively, the droplet size distribution can be selected to target the droplets to a compartment of the respiratory tract, such as the alveoli. In the case of aerosol delivery of pharmaceuticals, the droplets can have a diameter ranging from approximately 0.1 to 50 μm, preferably 1 to 25 μm, and more preferably 1 to 3 μm.

[0289] The aerosol droplets may be for delivery using a nebulizer (e.g., via the mouth) or a nasal spray. The aerosol formulation may optionally include a propellant and / or surfactant.

[0290] The formulation of pharmaceutical aerosols is routine for those skilled in the art; see, for example, Sciarra, J. in Remington's Pharmaceutical Sciences (supra). Medicaments can be formulated as solution aerosols, dry powders, emulsions, or dispersion or suspension aerosols of semisolid preparations. The aerosols may be delivered using any propellant system known to those skilled in the art. The aerosols may be applied to the upper or lower respiratory tract, or both, for example, via intranasal inhalation. The portion of the lung to which the medication is delivered may depend on the disorder. Compositions containing the vectors of the present invention may contain a humectant, particularly when intranasal delivery is to be used. This may help reduce or prevent mucosal drying and prevent membrane irritation. Suitable humectants include, for example, sorbitol, mineral oil, vegetable oil, and glycerol; emollients; membrane modulating agents; sweeteners; and combinations thereof. The compositions may contain a surfactant. Suitable surfactants include nonionic, anionic, and cationic surfactants. Examples of surfactants that can be used include, for example, polyoxyethylene derivatives of fatty acid partial esters of sorbitol anhydride, such as Tween 80, polyoxyl 40 stearate, polyoxyethylene 50 stearate, fusieates, bile salts, and octoxynol.

[0291] In some cases, subsequent administrations of viral / retroviral / lentiviral (e.g., SIV) vectors and / or non-viral nucleic acid molecules (e.g., plasmids, mRNA, or saRNA) may be administered after the initial administration. Administration may be, for example, at least 6 months, 8 months, 10 months, 1 year, or more after the initial administration. In some cases, the viral / retroviral / lentiviral (e.g., SIV) vectors and / or non-viral nucleic acid molecules (e.g., plasmids, mRNA, or saRNA) of the present invention may be administered at intervals of at least 6 months, 1 year, or longer. Preferably, administration is every 6 months, more preferably every year. The viral / retroviral / lentiviral (e.g., SIV) vectors and / or non-viral nucleic acid molecules (e.g., plasmids, mRNA, or saRNA) may be administered at the indicated intervals, for example, when the effect of the previous administration has diminished, and when additional administration does not exceed the therapeutic window and / or is not associated with one or more of the histopathological changes described herein.

[0292] Any two or more viral / retroviral / lentiviral (e.g., SIV) vectors and / or non-viral nucleic acid molecules (e.g., plasmids, mRNA, or saRNA) of the present invention can be administered separately, sequentially, or simultaneously. Thus, two or more viral / retroviral / lentiviral (e.g., SIV) vectors and / or non-viral nucleic acid molecules (e.g., plasmids, mRNA, or saRNA) can be administered separately, simultaneously, or sequentially, where at least one viral / retroviral / lentiviral (e.g., SIV) vector and / or non-viral nucleic acid molecule (e.g., plasmid, mRNA, or saRNA) is a viral / retroviral / lentiviral (e.g., SIV) vector and / or non-viral nucleic acid molecule (e.g., plasmid, mRNA, or saRNA) of the present invention. Specifically, two or more viral / retroviral / lentiviral (e.g., SIV) vectors and / or non-viral nucleic acid molecules (e.g., plasmids, mRNA, or saRNA) of the present invention can be administered in this manner. These two can be administered in the same or different compositions. In preferred cases, two viral / retroviral / lentiviral (eg, SIV) vectors and / or non-viral nucleic acid molecules (eg, plasmids, mRNA, or saRNA) may be delivered in the same composition.

[0293] Treatment according to the present invention preferably involves administering the medicament as an aerosol to a patient in need thereof. A breath-actuated nebulizer is preferably used when administering the aerosolized medicament to a patient, since the respiratory augmentation mechanism increases the proportion of aerosol generated during patient inhalation. Preferably, the aerosol is generated from a breath-actuated nebulizer device with a formulation capacity of 2 ml to 10 ml. Preferably, the breath-actuated nebulizer is capable of generating an aerosol of a formulation that is stable over the duration of aerosol delivery. "Stable aerosol generation" includes aerosols that possess the physical characteristics described above. It may also be preferable for the aerosol to be delivered to the patient at an aerosol delivery rate of 80 μl / min to 400 μl / min, as assessed under standard respiratory simulation conditions (sinusoidal breathing, 500 ml tidal volume, and 1:1 inhalation:exhalation ratio).

[0294] Animal models All existing animal models of PAP have drawbacks that limit their usefulness as models of PAP, particularly aPAP. The most common current animal model of PAP is the GM-CSF knockout (KO) mouse. However, GM-CSF KO mice do not express anti-GM-CSF autoantibodies, and it is currently unclear whether efficacy and / or toxicity may be affected by the presence of such autoantibodies. Several previous studies have shown that treatment of nonhuman primates with anti-GM-CSF antibodies can induce an aPAP-like phenotype. However, this model is too complex for initial proof-of-concept studies. Rasgrp1-deficient mice develop autoantibody-mediated PAP. However, this phenotype occurs only in older mice (approximately 12 months of age) and is associated with high mortality, making this model difficult to work with.

[0295] Thus, the present invention provides a novel animal model of PAP, particularly aPAP, that overcomes one or more of the problems associated with conventional PAP animal models. Specifically, the present invention developed a mouse model by passively immunizing mice with anti-mGM-CSF antibodies.

[0296] Specifically, the present invention provides a rodent model of aPAP, in which the rodent is passively immunized with an anti-GM-CSF antibody by intranasal administration.

[0297] Typically, rodent is mouse.The genetic background of mouse is not limited.Non-limiting examples of mouse strains that can be used in the mouse model of the present invention include C57 black 6 background mouse, wild-type mouse, or any mouse strain with relevant genetic modification, such as GM-CSF knockout mouse.

[0298] The anti-GM-CSF antibody used in the model of the present invention is not limited.When the model is a mouse model, mouse anti-GM-CSF antibody can typically be used.Non-limiting examples of mouse anti-GM-CSF antibodies that can be used include MMGM-CSF A7.39 and MMGM-CSF B2.6, which are described in Uyttenhove et al. (Eur.J.Immunol.2018.48:1883-1891), which is incorporated herein by reference in its entirety.Other non-limiting examples of anti-GM-CSF antibodies include GCA21, GCA7, and GCB59, which are described in Piccoli et al. (Nat.Comms.2015.6:7375), which is incorporated herein by reference in its entirety.

[0299] Typically, rodent models of aPAP achieve BALF anti-GM-CSF antibody concentrations of at least about 2 μg / mL, e.g., at least about 3 μg / mL, at least about 4 μg / mL, at least about 5 μg / mL, at least about 6 μg / mL, at least about 7 μg / mL, at least about 8 μg / mL, at least about 9 μg / mL, at least about 10 μg / mL, or more, preferably at least about 4 μg / mL. Non-limiting examples of BALF anti-GM-CSF antibody concentrations that can be achieved in rodent models of the present invention include about 1-10 μg / mL, e.g., about 2-7 μg / mL, about 4-6 μg / mL, or more, preferably about 4-6 μg / mL.

[0300] The present invention also provides a method for generating a rodent model of aPAP, comprising administering an anti-GM-CSF antibody to a rodent by intranasal administration. Any and all disclosures herein relating to the rodent model of the present invention apply equally and without reservation to the method for generating the rodent model of the present invention.

[0301] The rodent models of the present invention, particularly the mouse models, can be useful for studying aPAP as described herein. The rodent models of the present invention, particularly the mouse models, can be useful for studying drugs, cell products, biopharmaceuticals, or small molecules intended to treat aPAP, and optionally, compositions of the present invention, as described herein.

[0302] Preferably, anti-GM-CSF antibodies used in rodent models of the invention are prepared in pure form and / or at high concentrations prior to passive immunization. As a non-limiting example, one or more anti-GM-CSF antibodies used in rodent models of the invention can be prepared at a concentration of at least 600 μg / mL, e.g., at least 700 μg / mL, at least 750 μg / mL, at least 800 μg / mL, or more, e.g., about 820 μg / mL. Alternatively, or additionally, one or more anti-GM-CSF antibodies used in passive immunization in rodent models of the invention can have less than 0.1 ng of endotoxin per mg of antibody, e.g., less than 0.09 ng of endotoxin per mg of antibody, less than 0.08 ng of endotoxin per mg of antibody, less than 0.08 ng of endotoxin per mg of antibody, or less than 0.07 ng of endotoxin per mg of antibody, e.g., less than 0.06 ng of endotoxin per mg of antibody.

[0303] Anti-GM-CSF antibodies may be detectable in the BALF of a rodent model for about 1 to about 30 days, e.g., about 1 to about 20 days, about 5 to about 20 days, about 1 to about 15 days, about 5 to about 10 days, or about 1 to about 10 days, after passive immunization. Alternatively, or additionally, the concentration of anti-GM-CSF antibodies in the BALF of a rodent model may be at least about 5 ng / mL, e.g., at least about 6 ng / mL, at least about 7 ng / mL, at least about 8 ng / mL, at least about 9 ng / mL, at least about 10 ng / mL, at least about 11 ng / mL, at least about 12 ng / mL, at least about 13 ng / mL, at least about 14 ng / mL, at least about 15 ng / mL, or more.

[0304] A rodent model of PAP, particularly aPAP, can be generated by administering one or more anti-GM-CSF antibodies at a dose of about 1 μg / mouse to about 100 μg / mouse, for example, about 1 μg / mouse to about 50 μg / mouse, about 5 μg / mouse to about 50 μg / mouse, or about 10 μg / mouse to about 40 μg / mouse. When multiple anti-GM-CSF antibodies are administered to generate a model, the dose of each antibody to be administered can be determined independently. As a non-limiting example, a rodent model of PAP, particularly aPAP, can be generated by administering MMGM-CSF A7.39 and MMGM-CSF B2.6, optionally with MMGM-CSF A7.39 administered at a dose of 40 μg / mouse and MMGM-CSF B2.639 administered at a dose of 10 μg / mouse.

[0305] As shown herein, in some cases, repeated passive immunization may be required to maintain anti-GM-CSF antibody concentrations within the desired range in individual model subjects. As a non-limiting example, the one or more GM-CSF antibodies may be administered daily, every two days, every three days, every four days, every five days, every six days, every week, every ten days, every two weeks, or every month. In some preferred embodiments, the one or more anti-GM-CSF antibodies may be administered weekly. For example, a rodent model of PAP, particularly aPAP, can be generated by weekly administration of MMGM-CSF A7.39 and MMGM-CSF B2.6, optionally with MMGM-CSF A7.39 administered at a dose of 40 μg / mouse at each administration and MMGM-CSF B2.639 administered at a dose of 10 μg / mouse at each administration.

[0306] Repeated administration of antibodies can be administered for the duration required for the experiment being conducted on the model subject. For example, if the experiment is conducted over a 10-month period, the one or more anti-GM-CSF antibodies can be administered (e.g., weekly) for the 10-month experimental period.

[0307] Same arrangement Percent identity can be determined using any of a variety of sequence alignment methods, including, but not limited to, global, local, and hybrid methods, such as segment approaches. Protocols for determining percent identity are routine procedures within the skill of those in the art. Global methods align sequences from the beginning to the end of the molecule, summing the scores of individual residue pairs, and assigning gap penalties to determine the best alignment. Non-limiting examples of methods include CLUSTAL W (see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994)); and iterative refinement (see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J.MoI.Biol.823-838 (1996)). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences.Non-limiting methods include, for example, Matchbox, see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbs sampling, see, e.g., CE Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993); Align-M, see, e.g., Ivo Van Walle et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004).

[0308] Thus, percent sequence identity is determined by conventional methods. See, e.g., Altschul et al., Bull. Math. Bio. 48:603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment score using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) shown below (amino acids are shown in standard single-letter code).

[0309] The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, percent identity can be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids multiplied by 100. The calculation of percent sequence identity can also take into account the number of gaps and the length of each gap that need to be introduced to optimize the alignment of two or more sequences. The comparison of sequences and determination of percent identity between two or more sequences can be performed using specific mathematical algorithms, such as BLAST, which are familiar to those skilled in the art.

[0310] Alignment scores for determining sequence identity ARNDCQEGHILKMFPSTWYV A4 R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4 The percent identity is therefore: Total number of identical matches ____________________×100 [The length of the longer array is the length of the two arrays. To align the longer sequence plus the number of gaps introduced] It is calculated as:

[0311] Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions, or additions. These changes are preferably minor in nature, being conservative amino acid substitutions (described herein) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically from 1 to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.

[0312] In addition to the 20 standard amino acids, non-standard amino acids (e.g., 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) are substituted for amino acid residues in the polypeptides of the invention. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and unnatural amino acids are substituted for polypeptide amino acid residues. The polypeptides of the invention can also include non-naturally occurring amino acid residues.

[0313] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethyl cysteine, hydroxyethyl homo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods for incorporating non-naturally occurring amino acid residues into proteins are known in the art.For example, an in vitro system can be utilized in which nonsense mutations are suppressed using chemically aminoacylated suppressor tRNA.Methods for synthesizing amino acids and aminoacylation of tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations are carried out in a cell-free system containing Escherichia coli S30 extract and commercially available enzymes and other reagents. Proteins are 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 the second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). In the third method, E. coli cells are cultured in the absence of the natural amino acid to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).Non-naturally occurring amino acids are incorporated into polypeptides in place of their natural counterparts. See Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues are converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0314] A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids are substituted for amino acid residues in the polypeptides of the invention.

[0315] Essential amino acids in the polypeptides of the invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of the structure, determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of amino acids at putative contact sites. See, e.g., de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identity of essential amino acids can also be inferred from analysis of homology with related components of the polypeptides of the invention (e.g., translocation or protease components).

[0316] Multiple amino acid substitutions can be made and tested using known mutagenesis and screening methods, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions within a polypeptide, selecting functional polypeptides, and then screening the mutagenized polypeptides to determine the range of acceptable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0317] Multiple amino acid substitutions can be made and tested using known mutagenesis and screening methods, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions within a polypeptide, selecting functional polypeptides, and then screening the mutagenized polypeptides to determine the range of acceptable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0318] Sequence information SEQ ID NO: 1 hGM-CSF amino acid sequence (UniProt accession number P04141) SEQ ID NO: 2 hGM-CSF nucleic acid sequence (Genbank accession number M11220.1) SEQ ID NO: 3 mGM-CSF amino acid sequence (UniProt accession number P01587) SEQ ID NO: 4 mGM-CSF nucleic acid sequence (GenBank accession number AY950559.1) SEQ ID NO: 5 mGM-CSF transgene sequence contained in pIC017 and pIC098 SEQ ID NO: 6: Exemplary hCEF promoter SEQ ID NO: 7 Exemplary CMV promoter SEQ ID NO: 8 Exemplary EF1a promoter SEQ ID NO: 9 β-globin / IgG chimeric intron containing SIV RRE SEQ ID NO: 10 pIC017 hCEF mGMCSF Plasmid SEQ ID NO: 11 pIC098 CMV mGMCSF Plasmid SEQ ID NO: 12 Exemplary WPRE sequence SEQ ID NO: 13 Exemplary mifepristone-regulated promoter SEQ ID NO: 14. Exemplary transactivators for use with mifepristone-regulated promoters SEQ ID NO: 15 pDNA1 plasmid pSIV-2V-GMCSF (Figure 2B) SEQ ID NO: 16 pDNA2a plasmid pGM691 (Figure 2D) SEQ ID NO: 17 pDNA2a plasmid pGM297 (Figure 2E) SEQ ID NO: 18 pDNA2b plasmid pGM299 (Figure 2F) SEQ ID NO: 19 pDNA3a plasmid pGM301 (Figure 2G) SEQ ID NO: 20 pDNA3b plasmid pGM303 (Figure 2H) SEQ ID NO: 21 pDNA1* plasmid pSIV-2V-transactivator (Figure 2C) SEQ ID NO: 22 pDNA1 ta+ Plasmid pSIV-1V-GMCSF (Figure 2A) SEQ ID NO: 23 pDNA3 plasmid pMD2.G (Figure 2I) SEQ ID NO: 24 Codon-optimized gag-pol gene (from pGM691) SEQ ID NO: 25 Exemplary CAG promoter SEQ ID NO: 26: A preferred exemplary CMV promoter

[0319] SEQ ID NO: 1 hGM-CSF amino acid sequence (UniProt accession number P04141) MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE

[0320] Array No. 2 hGM-CSF nucleic acid sequence (Genbank accession number M11220.1) acacagagag aaaggctaaa gttctctgga ggatgtggct gcagagcctg ctgctcttgg gcactgtggc ctgcagcatc tctgcacccg cccgctcgcc cagccccagc acgcagccct gggagcatgt gaatgccatc caggaggccc ggcgtctcct gaacctgagt agagacactg ctgctgagat gaatgaaaca gtagaagtca tctcagaaat gtttgacctc caggagccga cctgcctaca gacccgcctg gagctgtaca agcagggcct gcggggcagc ctcaccaagc tcaagggccc cttgaccatg atggccagcc actacaagca gcactgccct ccaaccccgg aaacttcctg tgcaacccag attatcacct ttgaaagttt caaagagaac ctgaaggact ttctgcttgt catccccttt gactgctggg agccagtcca ggagtgagac cggccagatg aggctggcca agccggggag ctgctctctc atgaaacaag agctagaaac tcaggatggt catcttggag ggaccaaggg gtgggccaca gccatggtgg gagtggcctg gacctgccct gggcacactg accctgatac aggcatggca gaagaatggg aatattttat actgacagaa atcagtaata tttatatatt tatattttta aaatatttat ttatttattt atttaagttc atattccata tttattcaag atgttttacc gtaataatta ttattaaaaa tagcttctaa aaaaaaaaa

[0321] SEQ ID NO: 3 mGM-CSF amino acid sequence (UniProt accession number P01587) MWLQNLLFLGIVVYSLSAPTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPGQK

[0322] SEQ ID NO: 4 mGM-CSF amino acid sequence (Genbank accession number AY950559.1) atggctcacg agcggaaggc taaggtgctg cgcagaatgt ggctgcagaa cctgctgttc ctgggcatcg tggtgtacag cctgagcgcc cccaccagaa gccccatcac cgtgaccaga ccctggaagc acgtggaggc catcaaggaa gctctgaacc tgctggacga catgcccgtg accctgaacg aggaggtgga ggtggtgagc aacgagttta gctttaagaa gctgacctgc gtgcagaccc ggctgaagat cttcgagcag ggactgcggg gcaactttac caagctgaag ggagccctga acatgaccgc cagctactac cagacctact gccctcccac acccgagacc gactgtgaaa cccaggtgac cacctacgcc gactttatcg acagcctgaa gaccttcctg accgacatcc ccttcgagtg taagaagccc gtgcagaagt gactcgagcg g

[0323] SEQ ID NO: 5 mGM-CSF transgene sequence contained in pIC017 and pIC098 ctagccacca tgtggctgca gaacctgctg ttcctgggca ttgtggtgta cagcctgtct gcccctacaa gatcccctat cacagtgacc agaccttgga aacatgtgga agccatcaaa gaggccctga atctgctgga tgacatgcct gtgacactga atgaagaggt ggaagtggtg tccaatgagt tcagcttcaa gaaactgacc tgtgtgcaga ccaggctgaa gatttttgag cagggcctga gaggcaactt caccaagctg aaaggggctc tgaacatgac agccagctac taccagacct actgtcctcc tacacctgag acagactgtg aaaccccaagt gaccacctat gctgacttca ttgacagcct caagaccttc ctgacagaca tcccctttga gtgcaagaaa cctggccaga agtgagggcc

[0324] SEQ ID NO: 6: Exemplary hCEF promoter agatctgtta cataacttat ggtaaatggc ctgcctggct gactgcccaa tgacccctgc 60 ccaatgatgt caataatgat gtatgttccc atgtaatgcc aatagggact ttccattgat 120 gtcaatgggt ggagtattta tggtaactgc ccacttggca gtacatcaag tgtatcatat 180 gccaagtatg ccccctattg atgtcaatga tggtaaatgg cctgcctggc attatgccca 240 gtacatgacc ttatgggact ttcctacttg gcagtacatc tatgtattag tcattgctat 300 taccatggga attcactagt ggagaagagc atgcttgagg gctgagtgcc cctcagtggg 360 cagagagcac atggcccaca gtccctgaga agttgggggg aggggtgggc aattgaactg 420 gtgcctagag aaggtggggc ttgggtaaac tgggaaagtg atgtggtgta ctggctccac 480 ctttttcccc agggtggggg agaaccatat ataagtgcag tagtctctgt gaacattcaa 540 gcttctgcct tctccctcct gtgagtttgc tagc 574

[0325] SEQ ID NO:7 Exemplary CMV promoter ccgcggagat ctcaatattg gccattagcc atattattca ttggttatat agcataaatc 60 aatattggct attggccatt gcatacgttg tatctatatc ataatatgta catttatatt 120 ggctcatgtc caatatgacc gccatgttgg cattgattat tgactagtta ttaatagtaa 180 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg 240 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg 300 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta 360 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtcc gccccctatt 420 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttacgggac 480 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt 540 tggcagtaca ccaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac 600 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt 660 [[ID=A]]cgtaataacc ccgccccgtt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat 720 ataagcagag ctcgtttagt gaaccgtcag atcactagaa gctttattgc ggtagtttat 780 ... cacagttaaa ttgctaacgc agtcagtgct tctgacacaa cagtctcgaa cttaagctgc 840 agaagttggt cgtgaggcac tgggcaggct agc 873

[0326] SEQ ID NO: 8 Exemplary EF1a promoter agatccatat ccgcggcaat tttaaaagaa agggaggaat agggggacag acttcagcag 60 agagactaat taatataata acaacacaat tagaaataca acatttacaa accaaaattc 120 aaaaaatttt aaattttaga gccgcggaga tcccgtgagg ctccggtgcc cgtcagtggg 180 cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg 240 gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc 300 tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt 360 ttcgcaacgg gtttgccgcc agaacacagg ctagc 395

[0327] SEQ ID NO: 9 β-globin / IgG chimeric intron containing SIV RRE [ka] Underlined = β-globin / IgG chimeric intron Double underline = NotI restriction site Italics = SIV RRE sequence

[0328] SEQ ID NO: 10 pIC017 hCEF GMCSF Plasmid agatctgtta cataacttat ggtaaatggc ctgcctggct gactgcccaa tgacccctgc ccaatgatgt caataatgat gtatgttccc atgtaatgcc aatagggact ttccattgat gtcaatgggt ggagtattta tggtaactgc ccacttggca gtacatcaag tgtatcatat gccaagtatg ccccctattg atgtcaatga tggtaaatgg cctgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tatgtattag tcattgctat taccatggga attcactagt ggagaagagc atgcttgagg gctgagtgcc cctcagtggg cagagagcac atggcccaca gtccctgaga agttgggggg aggggtgggc aattgaactg gtgcctagag aaggtggggc ttgggtaaac tgggaaagtg atgtggtgta ctggctccac ctttttcccc agggtggggg agaaccatat ataagtgcag tagtctctgt gaacattcaa gcttctgcct tctccctcct gtgagtttgg taagtcactg actgtctatg cctgggaaag ggtgggcagg agatggggca gtgcaggaaa agtggcacta tgaaccctgc agccctagga atgcatctag acaattgtac taaccttctt ctctttcctc tcctgacagg ttggtgtaca gtagcttgct agccaccatg tggctgcaga acctgctgtt cctgggcatt gtggtgtaca gcctgtctgc ccctacaaga tcccctatca cagtgaccag accttggaaa catgtggaag ccatcaaaga ggccctgaat ctgctggatg acatgcctgt gacactgaat gaagaggtgg aagtggtgtc caatgagttc agcttcaaga aactgacctg tgtgcagacc aggctgaaga ttttgagca gggcctgaga ggcaacttca ccaagctga agggctctg aacatgacag ccagctacta ccagacctac tgtcctccta cacctgagac agactgtgaa acccaagtga ccacctatgc tgactcatt ccagcctca agaccttcct gagacatc ccctttgagt gcaagaaacc tggccagaag tgaggccct gtgccttcta gttgccagcc atctgttgtt tgcccctccc ctgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatt gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gagacaata gcaggcatgc agatcagcag ttcaacctgt tgatagtatg tactaagctc tcatgtttaa tgtactaagc tctcatgttt aatgaactaa accctcatgg ctaatgtact aagctcat ggctaatgta ctaagctctc atgtttcatg tactaagctc tcatgtttga acataaaat taatataaat cagcaactta atagcctct aaggttttaa gttttataag aaaaaaaga atatatagg cttttaaagg ttttaaggtt tcctaggtta tcctggtacc ttagaaaaac tcatccagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagtc ttttctgtaa tgaaggagaa aactcaccca ggcagttcca taggatggca agatcctggt atctgtctgc aattccaact cttccaacat caatacaacc tattaatttc ccctcatcaa aaataaggtt atcaagtgag aaatcaccat gagtgaccac tgaatctggt gagaatggca aaagcttatg catttctttc cagacttgtt caacaggcca gccattctc tcatcatcaa aatcactggc atcaaccaaa ccattattca ttcttgattg ggcctgagcc agtctaaata ctctatcaga gttaaaagga caattacaaa caggaatgga atgcaatctt ctcaggaaca ctgccagggc atcaacaata tttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc tgggatggca gtggtgagta accatgcatc atcaggagtt ctgataaaat gcttgatggt tggaagaggc ataaattcag tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacagaa cctttgccat gtttcagaaa caactctggg gcatctggct tcccatacaa tctatagatt gtggcacctg attgcccaac attatctcta gcccatttat acccatataa atcagcatcc atgttggaat ttaatcttgg cctggagcaa gaggtttctc tttgaatatg gctcatggat cccctcctat agtgagttgt attatactat gcagatatac tatgccaatg tttaattgtc aa

[0329] SEQ ID NO:11 pIC098 CMV GMCSF plasmid GGCATTGATT ATTGACTAGT TATTAATAGT AATCAATTAC GGGGTCATTA GTTCATAGCC CATATATGGA GTTCCGCGTT ACATAACTTA CGGTAAATGG CCCGCCTGGC TGACCGCCCA ACGACCCCCG CCCATTGACG TCAATAATGA CGTATGTTCC CATAGTAACG CCAATAGGGA CTTTCCATTG ACGTCAATGG GTGGAGTATT TACGGTAAAC TGCCCACTTG GCAGTACATC AAGTGTATCA TATGCCAAGT CCGCCCCCTA TTGACGTCAA TGACGGTAAA TGGCCCGCCT GGCATTATGC CCAGTACATG ACCTTACGGG ACTTTCCTAC TTGGCAGTAC ATCTACGTAT TAGTCATCGC TATTACCATG GTGATGCGGT TTTGGCAGTA CACCAATGGG CGTGGATAGC GGTTTGACTC ACGGGGATTT CCAAGTCTCC ACCCCATTGA CGTCAATGGG AGTTTGTTTT GGCACCAAAA TCAACGGGAC TTTCCAAAAT GTCGTAATAA CCCCGCCCCG TTGACGCAAA TGGGCGGTAG GCGTGTACGG TGGGAGGTCT ATATAAGCAG AGCTCGTTTA GTGAACCGTC AGATCACTAG AAGCTTTATT GCGGTAGTTT ATCACAGTTA AATTGCTAAC GCAGTCAGTG CTTCTGACAC AACAGTCTCG AACTTAAGCT GCAGAAGTTG GTCGTGAGGC ACTGGGCAGG TAAGTATCAA GGTTACAAGA CAGGTTTAAG GAGACCAATA GAAACTGGGC TTGTCGAGAC AGAGAAGACT CTTGCGTTTC TGATAGGCAC CTATTGGTCT TACTGACATC CACTTTGCCT TTCTCTCCAC AGGTGTCCAC TCCCAGTTCA ATTAAGCTC TTAAGGCTAG AGTACTTAAT ACGACTCACT ATAGGCTAGc tagccaccat gtggctgcag aacctgctgt tcctgggcat tgtggtgtac agcctgtctg cccctacaag atcccctatc acagtgacca gaccttggaa acatgtgaa gccatcaaag aggccctgaa tctgctggat gacatgcctg tgacactgaa tgaagaggtg gaagtggtgt ccaatgagtt cagcttcaag aaactgacct gtgtgcagac caggctgaag attttgagc agggcctgag aggcaacttc accaagctga aaggggctct gaacatgaca gccagctact accagaccta ctgtcctcct acacctgaga cagactgtga aacccaagtg accacctatg ctgacttcat tgacagcctc aagaccttcc tgacagacat cccctttgag tgcaagaaac ctggccagaa gtgagggccc tgtgccttct agttgccagc catctgttgt ttgcccctcc cctgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat tgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg cagatcagca gttcaacctg ttgatagtat gtactaagct ctcatgttta atgtactaag ctctcatgtt taatgaacta aaccctcatg gctaatgtac taagctctca tggctaatgt actaagctct catgtttcat gtactaagct ctcatgtttg aacaataaaa ttaatataaa tcagcaactt aaatagcctc taaggtttta agttttataa gaaaaaaaag aatatataag gcttttaaag gttttaaggt ttcctaggtt atcctggtac cttagaaaaa ctcatccagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagt cttttctgta atgaaggaga aaactcaccc aggcagttcc ataggatggc aagatcctgg tatctgtctg caattccaac tcttccaaca tcaatacaac ctattaattt cccctcatca aaaataaggt tatcaagtga gaaatcacca tgagtgacca ctgaatctgg tgagaatggc aaaagcttat gcatttcttt ccagacttgt tcaacaggcc agccatttct ctcatcatca aaatcactgg catcaaccaa accattattc attcttgatt gggcctgagc cagtctaaat actctatcag agttaaaagg acaattacaa acaggaatgg aatgcaatct tctcaggaac actgccaggg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc ctgggatggc agtggtgagt aaccatgcat catcaggagt tctgataaaa tgcttgatgg ttggaagagg cataaattca gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacaga acctttgcca tgtttcagaa acaactctgg ggcatctggc ttcccataca atctatagat tgtggcacct gattgcccaa cattatctct agcccattta tacccatata aatcagcatc catgttggaa tttaatcttg gcctggagca agaggtttct ctttgaatat ggctcatgga tcccctccta tagtgagttg tattatacta tgcagatata ctatgccaat gtttaattgt caa

[0330] SEQ ID NO: 12 Exemplary WPRE sequence gggcccaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat 60 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct 120 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag 180 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc 240 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc 300 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct 360 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg 420 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg 480 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg 540 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcaagct 600

[0331] SEQ ID NO: 13 Exemplary mifepristone-regulated promoter ACCGAGCTCTTACGCGGGTCGAAGCGGAGTACTGTCCTCCGAGTGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGTCGAGGGTCGAAGCGGAGTACTGTCCTCCGAGTGGAGTACTGTCCTCCGA GCGGAGTACTGTCCTCCGAGTCGACTCTAGAGGGTATATAATGGATCTCGAGATATCGGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGG GACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGCATTCCCCGTGTTAATTAACAGGTAAGTGTCTTCCTCCTGTTTCCTTCCCCTGCTATTCTGCTCAACCTTCCTATCAGAAACTGCAG TATCTGTATTTTTGCTAGCAGTAATACTAACGGTTCTTTTTTTCTCTTCACAGGCCACCAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCTGCAGATCGAAACGATGATAGATCCC

[0332] SEQ ID NO: 14. Exemplary transactivators for use with mifepristone-regulated promoters

[0333] SEQ ID NO: 15 pDNA1 plasmid pSIV-2V-GMCSF (Figure 2B)

[0334] Array number 16 pDNA2a plasmid pGM691 (Figure 2D) 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 gacttcctt gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct 1440 ctagcggcg cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc gggagggcc 1500 ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca gcctcggggc tgtccgcggg 1560 gggacggctg ccttcgggg 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 cagcagggaa acgcctgggt gcacgttcca ctgagcccta gaacactgaa 2220 tgcctgggtc aaagccgtgg aagagaagaa gtttggcgcc gagatcgtgc ccatgttcca 2280 ggctctgtct gagggctgca ccccttacga catcaaccag atgctgaacg tgctgggaga 2340 tcaccagggc gctctgcaga tcgtgaaaga gatcatcaac gaagaggctg cccagtggga 2400 cgtgacacat ccattgcctg ctggacctct gccagccgga caactgagag atcctagagg 2460 ctctgatatc gccggcacca ccagctctgt gcaagagcag ctggaatgga tctacaccgc 2520 caatcctaga gtggacgtgg gcgccatcta cagaagatgg atcatcctgg gcctgcagaa 2580 atgcgtgaag atgtacaacc ccgtgtccgt gctggacatc agacagggac ccaaagagcc 2640 cttcaaggac tacgtggacc ggttctataa ggccattaga gccgagcagg ccagcggcga 2700 agtgaagcag tggatgacag agagcctgct gatccagaac gccaatccag actgcaaagt 2760 gatcctgaaa ggcctgggca tgcaccccac actggaagag atgctgacag cctgtcaagg 2820 cgttggcggc ccttcttaca aagccaaagt gatggccgag atgatgcaga ccatgcagaa 2880 ccagaacatg gtgcagcaag gcggccctaa 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 cgagcaag 3480 atcctgaggg gcacaatcct gctggggcgcc acacctatca acatcatcgg cagaaatctg 3540 ctggcccctg ccggcgctag actggttatg ggacagctct ctgagaagat ccccgtgaca 3600 cccgtgaagc tgaaagaagg cgctagagga ccttgtgtgc gacagtggcc tctgagcaaa 3660 gagaattg aggccctgca agaaatctgt agccagctgg aacaagaggg caagatcagc 3720 agagttggcg gcgagaacgc ctacaatacc cctatcttct gcatcaagaa aaaggacaag 3780 agccagtggc ggatgctggt ggactttaga gagctgaaca aggctaccca ggacttcttc 3840 gaggtgcagc tgggaattcc tcatcctgcc ggcctgcgga agatgagaca gatcacagtg 3900 ctggatgtgg gcgacgccta ctacagcatc cctctggacc ccaacttcag aaagtacacc 3960 gccttcaa tccccaccgt gaaaatcaa ggccctggca tcagatacca gttcaactgc 4020 ctgcctcaag gctgggaaggg cagcccccacc attttcaga ataccgccgc cagcatcctg 4080 gaagaaatca agaaacct gcctgctctg accatcgtgc agtacatgga cgatctgtgg 4140 gtcggaagcc aagagaatga gcacacccac gacaagctgg tggaacagct gagacaaag 4200 ctgcaggcct ggggcctcga aacccctgag aagaaggtgc agaaagaacc tccttacgag 4260 tggatgggct acaagctgtg gcctcacaag tgggagctga gccggattca gctcgaagag 4320 aggacgagt ggaccgtgaa cgacatccag aaactcgtgg gcaagctgaa ttgggcagcc 4380 cagctgtatc ccggcctgag gaccaagaac atctgcaagc tgatccgggg aaagaagaac 4440 ctgctgggaac tggtcacatg gacacctgag gccgaggccg aatatgccga gaatgccgaa 4500 atcctgaaaa ccgagcaaga ggggacctac tacaagcctg gcattccaat cagagctgcc 4560 gtgcagaaac tggaaggcgg ccagtggtcc taccagttta agcaagaagg ccaggtcctg 4620 aaagtgggca agtacaccaa gcagaagaac acccacacca acgagctgag gacactggct 4680 ggcctggtcc agaaaatctg caaagaggcc ctggtcattt ggggcatcct gcctgttctg 4740 gaactgccca ttgagcggga agtgtgggaa cagtggtggg ccgattactg gcaagtgtct 4800 tggatccccg agtgggactt cgtgtctacc cctcctctgc tgaaactgtg gtacaccctg 4860 acaaaagagc ccattcctaa agaggacgtc tactacgttg acggcgcctg caaccggaac 4920 tccaaagaag gcaaggccgg ctacatcagc fotacggca agcagagagt ggaaaccctg 4980 gaaaacacca ccaaccagca ggccgagctg accgccatta agatggccct ggaagatagc 5040 ggccccaatg tgaacatcgt gaccgactct cagtacgcca tgggaatcct gacagcccag 5100 cctacacaga gcgatagccc tctggttgag cagatcattg ccctgatgat tcagaagcag 5160 caaatctacc tgcagtgggt gcccgctcac aaaggcatcg gcggaaacga agagatcgat 5220 aagctggtgt ccaagggaat cagacgggtg ctgttcctgg aaaagattga agaggcccaa 5280 gaggaacacg agcgctacca caacaactgg aagaatctgg ccgacaccta cggactgccc 5340 cagatcgtgg ccaaagaaat cgtggctatg tgccccaagt gtcagatcaa gggcgaacct 5400 gtgcacggcc aagtggatgc ttctcctggc acatggcaga tggactgtac ccacctggaa 5460 ggcaaagtgg tcatcgtggc tgtgcacgtg gcctccggct ttattgaggc cgaagtgatc 5520 cccagagaga caggcaaaga aaccgccaag ttcctgctga agatcctgtc cagatggccc 5580 atcacacagc tgcacaccga caacggccct aacttcacat ctcaagaggt ggccgccatc 5640 tgttggtggg gaaagattga gcacacaacc ggcattccct acaatccaca gagccagggc 5700 agcatcgagt ccatgaacaa gcagctcaaa gagattatcg gcaagatccg ggacgactgc 5760 footcacag aaacagccgt gctgatggcc tgtcacatcc aaacttcaa gcggaaaggc 5820 ggcatcggag gacagacatc tgccgagaga ctgatcaata tcatcaccac tcagctgggaa 5880 atccagcacc tccagaccaa gatccagaag attctgaact tccgggtgta ctaccgcgag 5940 ggcagagatc ctgtttggaa aggcccagca cagctgatct ggaaggcga aggtgccgtg 6000 gtgctgaagg atggctctga tctgaaggtg gtgcccagac ggaaggccaa gattatcaag 6060 gattacgagc ccaaacagcg cgtgggcaat gaaggcgacg ttgagggcac aagaggcagc 6120 gacaattgaa attcactcct caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg 6180 ccaatgccct ggctcacaaa taccactgag atctttttcc ctctgccaaa aattatgggg 6240 acatcatgaa gccccttgag catctgactt ctggctaata aaggaaaattt attttcattg 6300 caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc 6360 atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc catatgctgg 6420 ctgccatgaa caaaggttgg ctataaagag gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga aaagccttga cttgaggtta gatttttttt atttttgtt ttgtgttatt tttttcttta acatccctaa aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta ctcccagtca tagctgtccc tcttctctta tggagatccc 6660 tcgacctgca gcccaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 6720. tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc tcaattagtc agcaaccata gtccccgcccc 6960. taactccgcc catccccgcc ctaactccgc ccgttccgc cccctggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga 7020 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctaact tgtttattgc 7080. agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtcc 7200 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 7260 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 7320 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 7380 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 7440 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 7500 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 7560 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 7620 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 7680 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 7740 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 7800 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 7860 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 7920 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaga tcctttgatc 7980 tttctacgg gtctgacgc tcagtggaac gaaaaccc gttaaggat ttggtcatg 8040 agatttaca aaaggatct cacctagatc cttttaattt aaaaatgaag ttttaatca 8100 atctaagta tatgagta aacttggtct gagttaga aaactcatc gagcatcaa 8160 tgaaactgca atttattcat atcaggatta tcaataccat attttgaa aagccgtttc 8220 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcagatc ctggtatcgg 8280 tctgcgattc cgactcgtcc aacatcaata caactatta atttcccctc gtcaaaata 8340 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaacagc 8400 ttatgcattt ctttccagac ttgttcaca ggccagccat tacgctcgtc atcaaatca 8460 ctcgcatcaa ccaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga 8520 tcgctgttaa aaggacatt aaaacagga atcgaatgca accggcgcag gaacactgcc 8580 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt 8640 tttccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg 8700 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca 8760 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca 8820 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca 8880 tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga 8940 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat 9000 gatgatatat ttttatcttg tgcaatgtaa catcagagat tttgagacac aacaattggt 9060 cgac 9064

[0335] SEQ ID NO:17 pDNA2a plasmid pGM297 (Figure 2E) 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 ctttccta cagctcctgg 1680 gcaacgtgct gttattgtg ctgtctcatc attttgcaa agaattgctc gagactagtg 1740 acttggtgag taggcttcga gcctagttag aggactagga gaggccgtag ccgtactac 1800 tctgggcaag tagggcaggc ggtgggtacg caatggggc ggctacctca gcactaata 1860 gagacaatt agaccaattt gagaaaatac gactcgccc gaacgaag aaaagtacc 1920 aaattaaca ttatatatgg gcaggcagg agatggaggcg cttcggccctc catgagaggt 1980 tgttggagac agaggagggg tgtaaagaa tcatagaagt cctctacccc ctagaaccaa 2040 caggatcgga gggcttaaaa agtctgttca atcttgtgtg cgtactatat tgcttgcaca 2100 frequency agt frequency frequency cagtagcac agagquac cactgccatc 2160 tagtgaaaa agaaaaagt gcaacagaga helpctagtgg aaaaaaaaaaaaaaaaaaaagg 2220 gatagcagc gccacctggt ggcagtcaga attttccagc gcaacaaca ggaaatgcct 2280 gggtacatgt acccttgtca ccgcgcacct taaatgcgtg ggtaaaagca gtagaggaga 2340 aaaaatttgg agcagaaata gtacccatgt ttcaagccct atcagaaggc tgcacaccct 2400 atgacattaa tcagatgctt aatgtgctag gagatcatca aggggcatta caaatagtga 2460 aagagatcat taatgaagaa gcagcccagt gggatgtaac acaccacta cccgcaggac 2520 ccctaccagc aggacagctc agggaccctc gcggctcaga tatagcaggg accaccagct 2580 footcaaga acagttagaa tggatctata ctgctaaccc ccgggtagat gtaggtgcca 2640 tctaccggag atggattat ctaggacttc aaaagtgtgt caaaatgtac aacccagtat 2700 cagtcctaga cattaggcag ggacctaaag agcccttcaa ggattatgtg gacagatttt 2760 acaaggcaat tagagcagaa caagcctcag gggaagtgaa acaatggatg acagaatcat 2820 tactcattca aaatgctaat ccagattgta aggtcatcct gaagggccta ggaatgcacc 2880 ccacccttga agaaatgtta acggcttgtc aggggtagg aggcccaagc tacaaagcaa 2940 aagtaatggc agaaatgatg cagaccatgc aaaatcaaaa catggtgcag cagggaggtc 3000 caaaaagaca aagaccccca ctaagatgtt ataattgtgg aaaatttggc catatgcaaa 3060 gatagtcc ggaaccaagg aaaaaaaat gtctaaagtg tggaaattg ggacacctag 3120 caaagactg caggggacag gtgaattttt taggtatgg acggtggatg ggggcaaac 3180 cgagaaattt tcccgccgct actctggag cggaccgag tgcgcctcct cccaccgagcg 3240 gcaccacccc atacgaccca gcaagaagc tcctgcagca attgcagag aaagggaaac 3300 aactgaggga gcaaagagg aatccaccgg caatgaatcc ggattggacc gaggatatt 3360 ctttgaactc cctctttgga gagaccaat aagacagtg tatatagaag gggtccccat 3420 taaggcactg ctagacacag gggcagatga caccatatt aaagaaatg atttacaatt 3480 atcaggtcca tggagaccca aaattatagg gggcatagga ggaggcctta atgtaaaga 3540 ataacgac agggagta aaatagaga taaaattttg aggaggaaa tattgttagg 3600 agcaactccc attatata taggtagaaa ttgctggcc ccggcaggtg cccggttagt 3660 aatgggacaa ttatcagaa aaattcctgt cacacctgtc aaattgaagg aaggggctcg 3720 gggaccctgt gtaagacaat ggccctctc taagagaag attgaagctt tacaggaat 3780 atgttcccaa ttagagcagg aaggaaaaat cagtagagta ggaggagaa atgcataca 3840 taccccaata tttgcataa agagaagga caatcccag tggaggatgc tagtagactt 3900 taggagtta aaggcaa cccagattt ctttgaagtg cattaggga tacccaccc 3960 agcaggatta agaagatga agcagataac agttttagat gtaggagacg cctattattc 4020 cataccattg gatccaatt ttaggaata tactgctttt actattccca cagtgaataa 4080 tcagggaccc gggattaggt atcattcaa ctgtctcccg caagggtgga aaggatctcc 4140 tacaatcttc caaatacag cagcatccat tttggaggag aaaaagaa acttgccagc 4200 actaaccatt gtacaataca tggatgattt atgggtaggt tctcaagaaa atgacacac 4260 ccatgacaaa ttagtagaac agttagaac aaaattacaa gcctggggct tagaacccc 4320 agaaaagaag gtgcaaaag aaccacctta tgagtggatg ggatacaaac ttggcctca 4380 siaatgggaa ctaagcagaa tacaactgga ggaaaagat gatggactg tcaatgacat 4440 ccagaagtta gttgggaac taaattgggc agcacaatg tatccaggtc ttaggacca 4500 gatatatgc aagttatta gaggaaga aaatctgtta gagctagtga cttggacacc 4560 tgaggcagaa gctgaatg cagaaatgc agagattctt aaaacagaac aggaggaac 4620 ctattacaaa ccaggaatac ctattaggggc agcagtacag aaattggaag gaggacagtg 4680 gagttaccaa ttcaacaag aaggacaagt cttgaaagta ggaaataca ccaagcaaaa 4740 gaacaccat acaatgaac ttcgcacatt agctggttta gtgcagaaga tttgcaaga 4800 agctctagtt atttggggga tattaccagt tctgaacctc ccgatagaaa gagaggtag 4860 ggaacaatgg tgggcggatt actggcaggt aagctggatt cccgaatggg attttgtcag 4920 caccccacct ttgctcaac tatggtacac attaacaaa gaacccatac ccaaggagga 4980 cgtttactat gtagatgag catgcacag aaattcaaa gaaggaaag caggacaat 5040 ctcacaatac ggaaaacaga gagtagaaac attagaaac actaccaatc xxaaaaaa 5100 attackaaaatgg ctttggaga cagtggggcct atgtgaca tagtacaga 5160 ctctcaat gcaatgggaa ttttgacagc acaacccaca caagtgatt caccattagt 5220 agagcaatt atagccttaa tgatacaaaa gcaacaata tatttgcagt gggtaccagc 5280 acataagga atgagga atgaggagat agaataatta gtgagtaag gcattagag 5340 agttttattc ttagaaaaaa tagagaagc tchaagag catgaagat atcaataata 5400 ttggaaaaac ctagcagata catatgggct tccacaata gtagcaaag agatagtggc 5460 catgtgtcca aaatgtcaga taaagggaga accagtgcat ggacaagtgg atgcctcacc 5520 5580 tgtagccagt ggattcatag aagcagaagt catacctagg gaacaggaa aagaaacggc 5640 aaagtttcta ttaaaaatac tgagtagatg gcctataca cagttacaca cagacaatgg 5700 gcctaacttt acctcccaag aagtggcagc atatgttgg tggggaaaa ttgaacatac 5760 aacaggtata ccatataacc cccaatctca aggatcaata gaagcatga ashaacaat 5820 aaagagata attgggaaa taagagatga ttgccaatat acagagacag cagtactgat 5880 ggcttgccat attcacaatt ttaaaagaaa gggaggaata gggggacaga cttcagcaga 5940 gagactaatt aataataata caacacaatt agaaataca catttacaaa ccaaaattca aaaaatttta aattttagg tctactacag agaagggaga gaccctgtgt ggaaaggacc 6060 agcacaatta atctggaag gggaaggagc agtggtcctc aaggacgga gtgacctaaa ggttgtacca agaaggaag ctaaaattat taggattat gaacccaaac aaagagtggg 6240. 6240. 6240. 6240. 6240. 6240. gatattggat gagacaaaga aatttgaat ggaactatta tatgcatcag ctggcggccg cgaattcact agtgattccc gtttgtgcta gggttcttag gcttcttggg ggctgctgga 6360. actgcaatgg gagcagcggc gacagccctg acggtccagt ctcagcattt gcttgctggg 6420 atactgcagc agcagaaga tctgctggcg gctgtggagg ctcaacagca gatgttgaag ctgaccattt ggggtgttaa aaacctcaat gcccgcgtca cagcccttga gaagtaccta gaggatcagg cacgactaaa ctcctggggg tgcgcatgga aacaagtatg tcataccaca gtggagtggc cctggacaaa tcggactccg gattggcaaa atatgacttg gttggagtgg 6660 gaaagacaaa tagctgattt ggaaagcaac attacgagac aattagtgaa ggctagagaa 6720 caagaggaaa agaatctaga tgcctatcag aagttaacta gttggtcaga tttctggtct 6780 tggttcgatt tctcaaaatg gcttaacatt ttaaaaatgg gatttttagt aatagtagga 6840 தியுக்குக்குத்து taagattact ttacacagta tatggatgta tagtgagggt taggcaggga 6900 tatgttcctc tatctccaca gatccatatc caatcgaatt cccgcggccg caattcactc 6960 ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca 7020 aataccactg agatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg 7080 agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt 7140 ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg 7200 agtatttggt ttagagtttg gcaacatatg cccatatgct ggctgccatg aacaaaggtt 7260 ggctataaag aggtcatcag tatatgaaac agccccctgc tgtccattcc ttatccata 7320 gaaaagcctt gacttgaggt tagatttttt ttatattttg ttttgtgtta tttttttctt 7380 taacatccct aaaattttcc ttacatgttt tactagccag atttttcctc ctctcctgac 7440 tactcccagt catagctgtc cctcttctct tatggagatc cctcgacctg cagcccaagc 7500 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 7560 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 7620 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 7680 cggatccgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc 7740 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt 7800 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt 7860 ttggaggcct aggcttttgc aaaaagctaa cttgtttatt gcagcttata atggttacaa 7920 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg 7980 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ccgcttcctc gctcactgac 8040 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 8100 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 8160 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 8220 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 8280 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 8340 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 8400 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 8460 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 8520 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 8580 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga 8640 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 8700 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 8760 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 8820 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaatga agttttaaat caatctaaag tatatatgag 9000. 9000. 9000. 9000. 9000. 9000. 9000. 9000. 9000. 9000 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt 9180. snowflake 9180. snowflake 9180. snowflake tcaccatgag tgacgactga atccggtgag aatggcaaca gcttatgcat ttctttccag acttgttcaa caggccagcc attackcgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg tttttccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata 9540 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct 9600 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc 9660 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg 9720 ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc 9780 cttgtattac tgtttatgta agcagacagt tttattgttc atgatgatat atttttatct 9840 tgtgcaatgt aacatcagag attttgagac acaacaattg gtcgac 9886

[0336] SEQ ID NO: 18 pDNA2b plasmid pGM299 (Figure 2F) tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta 60 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 120 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg 180<x gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 240 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 300 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 360 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 420 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg 480 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 540 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 600 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc 660 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 720 tcgtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat 780 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc 840 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa 900 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac 960 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta 1020 aggtagagt acttaatacg actcactata ggctagccctc gagaattcga ttagcccct 1080 aggaccagaa gaaagaagat tgcttcgctt gatttggctc ctttacagca ccaatccata 1140 tccaccaagt ggggaaggga cggccagaca acgccgacga gccaggagaa ggtggagaca 1200 acagcaggat caattagag tcttggtaga aagactccaa gagcaggtgt atgcagttga 1260 ccgcctggct gacgaggctc aacacttggc tatacacag tgcctgacc ctcctcattc 1320 agcttagaat cactagtgaa ttcaccgtg gtaccttag agtcgaccg ggcggccgct 1380 tcgagcagac atgatagat acattgatga gtttggacaa accacacta gatgcagtg 1440 aaaaaaatgc tttattgtg aaatttgtga tgctattgct ttatttgtaa ccattataag 1500 ctgcaataaa caagttaaca acaacattg cattcatttt atgttcagg ttcaggggga 1560 gatgtgggag gttttttaaa gcaagtaaaa cctctacaa tgtggtaaaa tcgataagga 1620 tccgtcgacc aattgttgtg tctcaaatc tctgatgtta cattgcaca gataaaaata 1680 tatcatcatg aacataaaa ctgtctgctt acataacag tatacagg ggtgttatga 1740 gccatattca acgggaaacg tcttgctcta ggccgcgatt aaattccaac atggatgctg 1800 atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc 1860 gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg 1920 ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc 1980 cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc 2040 ccggaaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg 2100 atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta 2160 acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg 2220 atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa 2280 tgcataagct gttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg 2340 ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa 2400 tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt 2460 cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc 2520 agtttcattt gatgctcgat gagtttttct aactgtcaga ccaagtttac tcatatatac 2580 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg 2640 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 2700 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 2760 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 2820 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt 2880 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 2940 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 3000 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 3060 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 3120 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 3180 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 3240 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 3300 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 3360 ttgctcacat ggctcgacag atct 3384

[0337] SEQ ID NO: 19 pDNA3a plasmid pGM301 (Figure 2G) 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 agaattcgat tgccatggca 1740 acatatatcc agagagtaca gtgcatctca acatcactac tggttgttct caccacattg 1800 gtctcgtgtc agattcccag ggataggctc tctaacatag gggtcatagt cgatgaaggg 1860 aaatcactga agatagctgg atcccacgaa tcgaggtaca tagtactgag tctagttccg 1920 ggggtagact ttgagaatgg gtgcggaaca gcccaggtta tccagtaca gagcctactg 1980 aacaggctgt taatcccatt gagggatgcc ttagatctc aggaggctct gataactgtc 2040 accaatgata cgacacaaaa tgccggtgct ccccagtcga gattctcgg tgctgtgatt 2100 ggtactatcg cacttggagt gcgacatca gcacaaatca ccgcagggat tgcactagcc 2160 gaagcgagggg aggcaaag agacatagcg ctcatcaag atcgatgac aaaaacacac 2220 aagtctatag aactgctgca aaacgctgtg ggggaacaa ttctgctct aagacactc 2280 caggatttcg tgaatgatga gatcaaccc gcataagcg aattaggctg tgagactgct 2340 gccttaagac tgggtataaa attgacac cattactccg agctgttaac tgcgttcggc 2400 tcgaatttcg gaaccatcgg agagagagc ctcacgctgc agggctgtc ttcactttac 2460 tctgctaaca ttactgagat tatgaccaca atcaggacag ggcagtctaa catctatgat 2520 gtcatttata gagacagat gtgatagatg tggatctga gagatacatg 2580 gtcaccctgt ctgtgaagat ccctattctt tctgaagtcc caggtgtgct catacacaag 2640 gcatcatcta tttcttacaa catagacggg gaggaatggt atgtgactgt ccccagccat 2700 atactcagtc gtgcttcttt cttagggggt gcagacataa ccgattgtgt tgagtccaga 2760 ttgacctata tatgccccag ggatcccgca caactgatac ctgacagcca gcaaaagtgt 2820 atcctggggg acacaacaag gtgtcctgtc acaaaagttg tggacagcct tatccccaag 2880 tttgctttg tgaatggggg cgttgttgct aactgcatag catccacatg tacctgcggg 2940 acaggccgaa gaccaatcag tcaggatcgc tctaaaggtg tagtattcct aacccatgac 3000 aactgtggtc ttataggtgt caatggggta gaattgtatg ctaaccggag agggcacgat 3060 gccacttggg gggtccagaa cttgacagtc ggtcctgcaa ttgctatcag acccgttgat 3120 atttctctca accttgctga tgctacgaat ttcttgcaag actctaaggc tgagcttgag 3180 aaagcacgga aaatcctctc ggaggtaggt agatggtaca actcaagaga gactgtgatt 3240 acgatcatag tagttatggt cgtaatattg gtggtcatta tagtgatcat catcgtgctt 3300 tagatactca gaaggtgaaa tcactagtga attcactcct caggtgcagg ctgcctatca 3360 gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa taccactgag atctttttcc 3420 ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata 3480 aggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag 3540 gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc 3600 aacatatgcc catatgctgg ctgccatgaa caaaggttgg ctataaagag gtcatcagta 3660 tatgaaacag ccccctgctg tccattcctt attccataga aaagccttga cttgaggtta 3720 gattttttt atattttgtt ttgtgttatt tttttcttta acatccctaa aattttcctt 3780 acatgtttta ctagccagat ttttcctcct ctcctgacta ctcccagtca tagctgtccc 3840 tcttctctta tggagatccc tcgacctgca gcccaagctt ggcgtaatca tggtcatagc 3900 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca 3960 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct 4020 4080. cactcccgc tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttt gcagaggccg aggccgcctc 4200. ggcctctgag ctattccaga agtagtgagg aggcttttttt ggaggcctag gcttttgcaa 4260 aaagctaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc 4440 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 4620 gctcaagtca gaggtggcga aacccgacag gactaaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 4740 ttctccctc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 4800 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgacc cccgttcag cccgaccgct 4860 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttacgccac 4920 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 4980 tcttgaagtg gtggcctaac tacggctaca ctagagaac agtatttggt atctgcgctc 5040 tgctgaagcc agttaccttc ggaaaagag ttggtagctc ttgatccggc aaacaaacca 5100 cggctggtag cggtggtttt ttgttttgca agcagcagat tacgcgcaga aaaaaaggat 5160 ctcaagaga tcctttgatc tttctacgg ggtctgacgc tcagtggaac gaaaactcac 5220 gttaagggat ttggtcatg agattatca aaaggatct cacctagatc cttttaaatt 5280 aaaaatgag ttttaatca atctaagta tatgagta aacttggtct gagttaga 5340 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcataccat 5400 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga 5460 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta 5520 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat 5580 ccggtgagaa tggcaacagc ttatgcattt ctttccagac ttgttcaaca ggccagccat 5640 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct 5700 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca 5760 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt 5820 ctaatacctg gaatgctgtt tttccgggga tcgcagtggt gagtaaccat gcatcatcag 5880 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc 5940 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact 6000 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat 6060 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag 6120 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag 6180 cagacagttt tattgttcat gatgatatat ttttatcttg tgcaatgtaa catcagagat 6240 tttgagacac aacaattggt cgac 6264

[0338] SEQ ID NO: 20 pDNA3b plasmid pGM303 (Figure 2H) attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 60 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 120 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 180​​​​​​​​​​​​​​ 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 gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc 1620 taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc tggttattgt 1680 gctgtctcat cattttggca aagaattcct cgagcatgtg gtctgagtta aaaatcagga 1740 gcaacgacgg aggtgaagga ccagaggacg ccaacgaccc ccggggaaag ggggtgcaac 1800 acatccatat ccagccatct ctacctgttt atggacagag ggttagggat ggtgataggg 1860 gcaaacgtga ctcgtactgg tctacttctc ctagtggtag caccacaaaa ccagcatcag 1920 gttgggagag gtcaagtaaa gccgacacat ggttgctgat tctctcattc acccagtggg 1980 ctttgtcaat tgccacagtg atcatctgta tcataatttc tgctagacaa gggtatagta 2040 tgaaagagat ctcaatgact gtagaggcat tgaacatgag cagcagggag gtgaaagagt 2100 cacttaccag tctataagg caagaggtta tagcaagggc tgtcaacatt cagagctctg 2160 tgcaaaccgg aatcccagtc ttgttgaaca aaaacagcag ggatgtcatc cagatgattg 2220 ataagtcgtg cagcagacaa gagctcactc agcactgtga gagtacgatc gcagtccacc 2280 atgccgatgg aattgcccca cttgagccac atagtttctg gagatgccct gtcggagaac 2340 cgtatcttag ctcagatcct gaaatctcat tgctgcctgg tccgagcttg ttatctggtt 2400 ctacaacgat ctctggatgt gttaggctcc cttcactctc aattggcgag gcaatctatg 2460 cctattcatc aaatctcatt acacaaggtt gtgctgacat agggaaatca tatcaggtcc 2520 tgcagctagg gtacatatca ctcaattcag atatgttccc tgatcttaac cccgtagtgt 2580 cccacactta tgacatcaac gaatcgga aatcatgctc tgtggtggca accgggacta 2640 ggggttatca gctttgctcc atgccgactg tagacgaaag aaccgactac tctagtgatg 2700 gtattgagga tctggtcctt gatgtcctgg atctcaaagg gagaaag tctcaccggt 2760 atcgcaacag cgaggtagat cttgatcacc cgttctctgc actatacccc agtgtaggca 2820 acggcattgc aacagaaggc tcattgatat ttcttgggta tggtggacta accacccctc 2880 tgcagggtga tacaaaatgt aggacccaag gatgccaaca ggtgtcgcaa gacacatgca 2940 atgaggctct gaaaattaca tggctaggag ggaaacaggt ggtcagcgtg atcatccagg 3000 tcaatgacta tctctcagag aggccaaaga taagagtcac aaccattcca atcactcaaa 3060 actatctcgg ggcggaaggt agattattaa aattgggtga tcgggtgtac atctatacaa 3120 gatcatcagg ctggcactct caactgcaga taggagtact tgatgtcagc caccctttga 3180 ctatcaactg gacacctcat gaagccttgt ctagaccagg aaataaagag tgcaattggt 3240 acaataagtg tccgaaggaa tgcatatcag gcgtatacac tgatgcttat ccattgccc 3300 ctgatgcagc taacgtcgct accgtcacgc tatatgccaa tacatcgcgt gtcaacccaa 3360 caatcatgta ttctaacact actaacatta taaatatgtt aaggataaag gatgttcaat 3420 tagaggctgc atataccacg acatcgtgta tcacgcattt tggtaaaggc tactgctttc 3480 acatcatcga gatcaatcag aagagcctga ataccttaca gccgatgctc tttaagacta 3540 gcatccctaa attatgcaag gccgagtctt aagcggccgc gcatgcgaat tcactcctca 3600 ggtgcaggct gcctatcaga aggtggtggc tggtgtggcc aatgccctgg ctcacaaata 3660 ccactgagat ctttttccct ctgccaaaaa ttatggggac atcatgaagc cccttgagca 3720 tctgacttct ggctaataaa ggaaatttat tttcattgca atagtgtgtt ggaatttttt 3780 gtgtctctca ctcggaagga catatgggag ggcaaatcat ttaaaacatc agaatgagta 3840 tttggtttag agtttggcaa catatgccca tatgctggct gccatgaaca aaggttggct 3900 ataaagaggt catcagtata tgaaacagcc ccctgctgtc tattccttat tccatagaaa 3960 agccttgact tgaggttaga ttttttttat attttgtttt gtgttatttt tttctttaac 4020 atccctaaaa ttttccttac atgttttact agccagattt ttcctcctct cctgactact 4080 cccagtcata gctgtccctc ttctcttatg gagatccctc gacctgcagc ccaagcttgg 4140 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca 4200 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 4260 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagcgga 4320 tccgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct 4380 aactccgcccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc 4440 agaggccgag gccgctcgg cctctgagct attccagaag tagtgaggag gcttttttgg 4500 aggcctaggc ttttgcaaaa agctaacttg tttattgcag cttataatgg ttacaaataa 4560 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt 4620 ttgtccaaac tcatcaatgt atcttatcat gtctgtccgc ttcctcgctc actgactcgc 4680 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 4740 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 4800 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 4860 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 4920 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 4980 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 5040 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 5100 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 5160 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 5220 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag 5280 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 5340 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 5400 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 5460 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 5520 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 5580 cttggtctga cagttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat 5640 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac 5700 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa 5760 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac 5820 catgagtgac gactgaatcc ggtgagaatg gcaacagctt atgcatttct ttccagactt 5880 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat 5940 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac 6000 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac 6060 ctgaatcagg atattcttct aatacctgga atgctgtttt tccggggatc gcagtggtga 6120 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt 6180 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc 6240 catgtttcag aaacaactct ggcgcatcgg gcttccata caatcgatag attgtcgcac 6300 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg 6360 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg 6420 tattactgtt tatgtaagca gacagtttta ttgttcatga tgatatattt ttatcttgtg 6480 caatgtaaca tcagagattt tgagacacaa caattggtcg ac 6522

[0339] SEQ ID NO: 21 pDNA1* plasmid pSIV-2V-transactivator (Figure 2C)

[0340] SEQ ID NO: 22 pDNA1 ta+ Plasmid pSIV-1V-GMCSF (Figure 2A)

[0341] SEQ ID NO: 23 pDNA3 plasmid pMD2.G (Figure 2I)

[0342] Array number 24 codon-optimized gag-pol gene 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 ggcctcctct gagatgctac 1200 aactgcggca agttcggcca catgcagaga cagtgtcctg agcctaggaa aacaaaaatgt 1260 ctaaagtgtg gaaaattggg acacctagca aaagactgca ggggacaggt gaatttttta 1320 gggtatggac ggtggatggg ggcaaaaccg agaaattttc ccgccgctac tcttggagcg 1380 gaaccgagtg cgcctcctcc accgagcggc accaccccat acgacccagc aaagaagctc 1440 ctgcagcaat atgcagagaa agggaaaca ctgagggagc aaaagagaa tccaccggca 1500 atgaatccgg attggaccga gggatattct ttgaactccc tctttggaga agaccaataa 1560 agaccgtgta catcgagggc gtgcccatca aggctctgct ggatacaggc gccgacgaca 1620 ccatcatcaa agagaacgac ctgcagctga gcggcccttg gaggcctaag atcattggag 1680 gaatcgggggg aggcctgaac gtcaaagagt acaacgaccg ggaagtgaag atcgagca 1740 agatcctgag gggcacaatc ctgctgggcg ccacacctat caacatcatc ggcagaaatc 1800 tgctggcccc tgccggcgct agactggtta tgggacagct ctctgagaag atccccgtga 1860 cacccgtgaa gctgaaagaa ggcgctagag gaccttgtgt gcgacagtgg cctctgagca 1920 aagagaagat tgaggccctg caagaaatct gtagccagct ggaacaagag ggcaagatca 1980 gcagagttgg cggcgagaac gcctacaata cccctatctt ctgcatcaag aaaaaggaca 2040 agagccagtg gcggatgctg gtggacttta gagagctgaa caaggctacc 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 ggaaaagatt 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 acggaaggcc aagattatca 4320 aggattacga gcccaaacag cgcgtgggca atgaaggcga cgttgagggc acaagaggca 4380 gcgacaattg a 4391

[0343] SEQ ID NO: 25 Exemplary CAG Promoter 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 gagccacc 1738

[0344] SEQ ID NO: 26 Exemplary CMV promoter gcattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc 60 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 120 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 180 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 240 agtgtatcat atgccaagtc cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 300 gcattatgcc cagtacatga ccttacggga ctttcctact tggcagtaca tctacgtatt 360 agtcatcgct attaccatg 379

[0345] Example The present invention will now be described with reference to the following examples. These 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. The examples can therefore be considered as constituent elements of the invention, and the individual aspects described therein can be considered to be disclosed independently or in any combination. [Example]

[0346] Prolonged and / or high levels of mGM-CSF expression are associated with significant histopathological changes. GM-CSF has several functions within the intrinsic immune / inflammatory cascade, including differentiation of granulocytes (neutrophils, eosinophils, and basophils) and monocytes / macrophages from precursors. As such, it is likely to be a tightly regulated protein and therefore has a well-defined efficacy / toxicity window.

[0347] Untreated PAP mice showed mild pulmonary inflammatory changes and evidence of alveolar thickening, as well as a mild phenotype in the liver, all of which are likely related to the lack of GM-CSF expression in these knockout mice. Transduction of a control vector expressing Glux did not alter these baseline changes.

[0348] The lungs of GM-CSF knockout mice were infected with increasing doses of rSIV.F / HN-mGM-CSF (1 × 10 7 ~92×10 7 Two months after treatment, histopathology was performed on wild-type mice (WT), untreated knockout mice (PAP), and mice treated with a control vector (Glux, 24 × 10 7 The results were compared with knockout mice treated with TU / mouse (n = 3-6 mice / group). Analysis was performed blinded and scored using a semiquantitative scoring system. Scores are defined as follows: - absent, - / + equivocal, ++ mild, +++ moderate, ++++ severe. PAM = alveolar macrophages.

[0349] [Table 2]

[0350] Based on the long-term expression characteristics of the rSIV.F / HN platform technology, we next 7 We evaluated whether long-term mGM-CSF expression in mice receiving TU / mouse leads to histopathological changes. Lungs of GM-CSF knockout mice were transfected with rSIV.F / HN-mGM-CSF or Glux control lentivirus (1 × 10 7 Mice were treated with GM-CSF (TU / mouse) (n = 3-10 mice / group). Histopathology was compared between GM-CSF- and Glux-treated mice at time points ranging from 1 week to 9 months after treatment. Analysis was performed blinded and scored using a semiquantitative scoring system. Scores were defined as follows: - absent, - / + equivocal, ++ mild, +++ moderate, and ++++ severe. PAM = alveolar macrophages. In the former group, we observed progressively more severe histopathological changes in the lungs 6-9 months after treatment, including tissue consolidation, inflammation, and increased numbers of lung macrophages. In addition, at 9 months, we also observed accumulation of inflammatory cells and vasodilation in the kidneys.

[0351] [Table 3]

[0352] To further define the suitable efficacy / toxicity window in this model, histopathological analysis was performed on 1 x 10 5 and 1 × 10 6 In animals treated with TU / mouse (n = 4-7 per group), the improvement was extended to 11 months. As noted above, mice treated with these relatively low doses showed significant improvements in PAP biomarkers, even when vector-induced mGM-CSF levels were below the lower limit of detection for this assay. After 11 months of treatment, analysis was performed blinded and assessed using a semiquantitative scoring system. Scores were defined as follows: - absent, - / + equivocal, ++ mild, +++ moderate, and ++++ severe. PAM = alveolar macrophages.

[0353] Histopathological analysis at this time point was performed on 1 × 10 5 or 1×10 6 TU / mice showed no or moderate histopathology, respectively (Table 3). We were therefore able to define an efficacy / toxicity window in this mouse model.

[0354] [Table 4] [Example]

[0355] Non-viral GM-CSF gene therapy results in sufficient mGM-CSF expression to alleviate the PAP phenotype, and expression is stopped before toxicity is observed. Mice were treated with an exemplary non-viral expression plasmid (phCEFI-GM-CSF) complexed with GL67A, and GM-CSF expression was measured 1, 2, and 6 months after a single treatment. As shown in Figure 3, GM-CSF expression was no longer detectable at 6 months after treatment. 1 x 10 6The effective GM-CSF expression levels from the lentiviral-treated group in TU / mice were used for comparison. This experiment demonstrates that non-viral GM-CSF gene therapy can be used to deliver and express mGM-CSF at levels sufficient to alleviate the PAP phenotype, and that expression is halted before long-term GM-CSF expression can cause toxicity.

[0356] The possibility of sustained treatment effects after a single GL67A / mGM-CSF pDNA dose was further investigated. GM-CSF knockout mice were treated with the GL67A-mGM-CSF pDNA complex at a dose of 80 μg / mouse. Untreated WT mice were included as a reference. Animals were sacrificed 1 to 10 months after transfection, and mGM-CSF expression was quantified in lung homogenates. As shown in Figure 4A, the presence of mGM-CSF was observed at 1 and 3 months, but at 10 months, mGM-CSF levels were indistinguishable from those in wild-type controls.

[0357] The effect of mGM-CSF expression on multiple biomarkers of PAP was analyzed. As shown in Figure 4B, BALF turbidity remained observably reduced compared to untransfected controls, even at 10 months, when mGM-CSF was no longer detectable in lung homogenates. Similarly, surfactant protein D (SP-D) concentrations in lung homogenates (shown in Figure 4C) and BALF (Figure 4D) remained observably reduced over the time course of the experiment, even when mGM-CSF expression in lung homogenates was no longer detectable. Finally, surfactant deposition in alveoli was quantified as the percentage of PAS-positive alveoli. As shown in Figure 4E, little surfactant deposition was observed over the entire time course after a single treatment with GL67A / mGM-CSF pDNA, even several months after mGM-CSF was no longer detectable. [Example]

[0358] Non-viral GM-CSF gene therapy is long-lasting and non-toxic The experiment in Example 2 was repeated to investigate the occurrence of histopathological changes in GM-CSF knockout mice treated with a single GL67A / pDNA dose (80 μg / mouse) and observe any changes over a 10-month period. Animals were sacrificed 1 to 10 months after transfection with GL67A / pDNA. Histopathological analysi...

Claims

1. A granulocyte-macrophage colony-stimulating factor (GM-CSF) gene therapy agent for use in the treatment of pulmonary alveolar proteinosis (PAP), said agent transiently expressing GM-CSF in a patient.

2. The therapeutic agent for use according to claim 1, wherein the transient GM-CSF protein expression is expression for 6 months or less, preferably 4 months or less, more preferably 3 months or less.

3. The treatment comprises: (a) Bronchoalveolar lavage fluid (BALF) turbidity; (b) surfactant protein D (SF-D) concentration in the lung; (c) SF-D concentration in BALF; (d) surfactant deposition in the lungs; and / or (e) pulmonary pathology, optionally selected from (i) pulmonary opacity, (ii) pulmonary edema, and / or (iii) pulmonary consolidation; or reducing one or more PAP biomarkers selected from and / or said treatment optionally comprises measuring (i) vital capacity (VC); (ii) forced vital capacity (FVC); and / or (iii) forced expiratory volume (FEV), in particular FEV1; (iv) arterial oxygen tension (Pa,O ２ ); (v) alveolar-arterial oxygen partial pressure gradient (PA-a,O ２ (vi) Peak Metabolic Equivalents (Peak METS), and / or (vii) Six Minute Walking Distance (6MWD), preferably PA-a,O ２ 3. The therapeutic agent for use according to claim 1 or 2, which increases lung function selected from increasing lung function.

4. The treatment is not associated with one or more histopathological changes in the patient, and the one or more histopathological changes are optionally: (a) one or more histopathological changes in the lung, optionally including distortion of lung architecture, pulmonary inflammatory cell infiltration exceeding the PAP phenotype, increased alveolar wall thickness, pulmonary alveolar microlithiasis (PAM) alveoli, PAM bronchi, presence of neutrophils in the bronchi, consolidation, presence of giant cells, eosinophilic material, and / or edema; (b) one or more histopathological changes in the liver, optionally including inflammatory cell infiltration exceeding the PAP phenotype, inflammation in the portal tract, sinusoidal dilation and congestion, and / or vascular dilation and congestion; (c) one or more histopathological changes in the kidney, optionally inflammatory cell infiltration beyond the PAP phenotype, vascular dilation, fibrosis, eosinophilic material, and / or cysts; and / or (d) one or more histopathological changes in the spleen, optionally the presence of clusters of megakaryocytes and / or macrophages The therapeutic agent for use according to any one of claims 1 to 3, selected from the group consisting of:

5. The therapeutic agent is (a) a non-viral nucleic acid molecule encoding GM-CSF, and a lipid carrier; or (b) a viral vector or a non-viral nucleic acid molecule comprising a GM-CSF transgene operably linked to an inducible promoter. The therapeutic agent for use according to any one of claims 1 to 4, comprising:

6. The therapeutic agent for use according to claim 5, wherein the non-viral nucleic acid molecule is a plasmid comprising a GM-CSF transgene operably linked to a promoter.

7. 7. The therapeutic agent for use according to claim 6, wherein (i) the GM-CSF transgene; (ii) the promoter; or (iii) the GM-CSF transgene and the promoter contain 10 or fewer CpG dinucleotides or no CpG dinucleotides.

8. 8. The therapeutic agent for use according to claim 6 or 7, wherein the plasmid comprises the GM-CSF transgene operably linked to a promoter 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, the plasmid comprises a hybrid human CMV enhancer / EF1a (hCEF) promoter.

9. The therapeutic agent for use according to claim 5, wherein the non-viral nucleic acid molecule is mRNA or self-amplifying RNA (saRNA) encoding GM-CSF.

10. (a) the therapeutic agent is an mRNA comprising pseudouridine (ψ-UTP), Cap1, and / or a poly(A) tail of about 10-100 adenosine nucleotides, optionally wherein the mRNA is about 0.5 kb to about 5 kb in length; (b) The therapeutic agent for use according to claim 9, wherein the therapeutic agent is a saRNA comprising ψ-UTP, Cap1, and / or a poly(A) tail of about 10 to 100 adenosine nucleotides, and optionally the saRNA is about 9 kb to about 12 kb in length.

11. (a) the lipid carrier is a lipid nanoparticle, preferably a liposome; (b) the lipid carrier comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids; and / or (c) The therapeutic agent for use according to any one of claims 5 to 10, wherein the lipid carrier is GL67A.

12. The therapeutic agent for use according to claim 5, wherein the viral vector is a lentiviral or retroviral vector.

13. The lentiviral or retroviral vector comprises: (a) pseudotyped with (i) the hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus, preferably Sendai virus, or (ii) the G glycoprotein from vesicular stomatitis virus (G-VSV); and / or (b) a lentiviral vector selected from the group consisting of simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV) vector, feline immunodeficiency virus (FIV) vector, equine infectious anemia virus (EIAV) vector, and visna / maedi virus vector, preferably an SIV vector.

14. The inducible promoter is (a) (i) a steroid-regulated promoter, preferably a mifepristone-regulated promoter; or (ii) a chemically-regulated promoter; and / or (b) (i) the transgene operably linked to an inducible promoter and the transactivator of the inducible promoter are contained in (i) the same lentiviral or retroviral vector, or (ii) separate lentiviral or retroviral vectors.

15. 15. The therapeutic agent for use according to any one of claims 1 to 14, formulated for administration to the lungs; optionally, said administration is by intratracheal or intranasal instillation, aerosol delivery, nebulization, intravenous injection, or direct injection into the lungs.

16. The therapeutic agent for use according to any one of claims 1 to 15, wherein the PAP is autoimmune PAP (aPAP).

17. A method of treating PAP, comprising administering a therapeutically effective amount of a GM-CSF gene therapy agent to a patient in need thereof.

18. Use of a GM-CSF gene therapy agent in the manufacture of a medicament for the treatment of PAP.

19. (a) a non-viral nucleic acid molecule encoding GM-CSF, and a lipid carrier; or (b) a viral vector containing a GM-CSF transgene operably linked to an inducible promoter. A composition comprising: The composition is formulated for administration to the lung such that, upon administration, the non-viral nucleic acid molecule or viral vector is capable of transiently expressing GM-CSF in cells of the lung.

20. (a) the non-viral vector is a plasmid according to any one of claims 6 to 8; or (b) the non-viral vector is an mRNA or saRNA according to claim 9 or 10; 20. The composition of claim 19, wherein the lipid carrier is as defined in claim 11.

21. A rodent model of aPAP, wherein the rodent is passively immunized with anti-GM-CSF antibodies by intranasal administration.

22. (a) the rodent is a mouse, optionally a mouse having a C57 Black 6 background, a wild-type mouse, or a GM-CSF knockout mouse; (b) the anti-GM-CSF antibody is a murine anti-GM-CSF antibody; and / or (c) The rodent model of claim 21, wherein the model achieves a BALF anti-GM-CSF antibody concentration of about 4-6 μg / mL or more.

23. A method for generating a rodent model of aPAP, comprising administering an anti-GM-CSF antibody to a rodent by intranasal administration.

24. (a) the rodent is a mouse, optionally a mouse having a C57 Black 6 background, a wild-type mouse, or a GM-CSF knockout mouse; and / or (b) the anti-GM-CSF antibody is a murine anti-GM-CSF antibody; and / or (c) the model achieves a BALF anti-GM-CSF antibody concentration of about 4-6 μg / mL or more.

25. (a) To study aPAP; (b) Use of a rodent model according to claim 21 or 22 for studying drugs, cell products, biopharmaceuticals or small molecules intended for the treatment of aPAP, optionally for studying compositions as defined in claim 19 or 20.

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