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Pathotropic targeted gene delivery system for cancer and other disordersRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Whole Live Micro-organism, Cell, Or Virus Containing, Genetically Modified Micro-organism, Cell, Or Virus (e.g., Transformed, Fused, Hybrid, Etc.)Pathotropic targeted gene delivery system for cancer and other disorders description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070178066, Pathotropic targeted gene delivery system for cancer and other disorders. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE [0001] This application is a continuation-in-part of U.S. application Ser. No. 10/829,926, filed Apr. 21, 2004, which claims priority from U.S. Provisional Application Ser. No. 60/464,571, filed Apr. 21, 2003, which is incorporated herein by reference. TECHNICAL FIELD [0002] The present invention relates generally to methods and compositions for treating neoplastic disorders. Further, the invention relates to methods and systems for producing therapeutically effective vectors. BACKGROUND OF THE INVENTION [0003] Approximately 70% of all gene therapy protocols are aimed at treating metastatic cancer. The majority of active protocols involve some form of cancer immunotherapy via cell-based gene transfer of cytokines or tumor antigens, while others involve the intratumoral delivery of oncolytic viruses or vectors bearing prodrugs, chemoprotective agents, antisense constructs, or tumor suppressor genes. However, the major unresolved problem that has hindered the development and deployment of effective cancer gene therapy is that of inefficient delivery to target cells in vivo, a problem that obviates and precludes many direct therapeutic approaches (Tseng and Mulligan, Surg. Oncol. Clin. N. Am. 11:537-569, 2002). In this regard, the advent of pathotropic targeting launches a new paradigm in cancer gene therapy. By targeting the histopathology of the lesion--rather than the cancer cells per se--to optimize the effective vector concentration at metastatic sites, the safety and the efficacy of the circulating gene therapy vector was increased dramatically in preclinical studies (Gordon et al., Cancer Res. 60:3343-3347, 2000; Gordon et al., Hum. Gene Ther. 12:193-204, 2001). Further enhanced by the inherent properties of the murine leukemia virus-based vector (which selectively transduces dividing cells) and the strategic specificity of a cell cycle control gene which exhibits tumoricidal and anti-angiogenic activities (Gordon et al., Hum. Gene Ther. 12:193-204, 2001), the preclinical and clinical performance of the pathotropic vector establishes the potential for systemic delivery of genetic medicine for the physiologic surveillance and treatment of primary, remote, metastatic, and occult cancers. [0004] Improved vectors, systems for producing the improved vectors, and treatment regimens for administering such vectors, are desired so that targeted delivery systems can be employed in a clinical setting. SUMMARY OF THE INVENTION [0005] This disclosure relates to "targeted" viral and non-viral particles, including retroviral vector particles, adenoviral vect particles, adeno-associated virus vector particles, Herpes Virus vector particles, and pseudotyped viruses such as with the vesicular stomatitis virus G-protein (VSV-G), and to non-viral vectors that contain a viral protein as part of a virosome or other proteoliposomal gene transfer vector. [0006] Also provided are novel retroviral expression systems for the generation of targeted viral particles, the use of transiently transfected human producer cells to produce the particles, a manufacturing process for large scale production of the viral particles, and methods for collecting and storing targeted viral vectors. [0007] In one embodiment, a method for producing a targeted delivery vector is provided. The method includes transiently transfecting a producer cell with 1) a first plasmid comprising a nucleic acid sequence encoding the 4070A amphotropic envelope protein modified to contain a collagen binding domain; 2) a second plasmid comprising i) a nucleic acid sequence operably linked to a promoter, wherein the sequence encodes a viral gag-pol polypeptide; ii) a nucleic acid sequence operably linked to a promoter, wherein the sequence encodes a polypeptide that confers drug resistance on the producer cell; and iii) an SV40 origin of replication; 3) a third plasmid comprising i) a heterologous nucleic acid sequence operably linked to a promoter, wherein the sequence encodes a diagnostic or therapeutic polypeptide; ii) 5' and 3' long terminal repeat sequences; iii) a .PSI. retroviral packaging sequence; iv) a CMV promoter upstream of the 5' LTR; v) a nucleic acid sequence operably linked to a promoter, wherein the sequence encodes a polypeptide that confers drug resistance on the producer cell; vi) an SV40 origin of replication. The producer cell is a human cell that expresses SV40 large T antigen. In ones aspect, the producer cell is a 293T cell. [0008] The method further includes culturing the transfected producer cells of under conditions that allow the targeted delivery vector to be produced in the supernatant of the culture and isolating and introducing the supernatant in to a closed loop manifold system for collecting the vector. An exemplary closed loop manifold system is set forth in FIG. 19A and FIG. 19B. In one embodiment, the targeted delivery vector is a viral particle. In another embodiment, the targeted delivery vector is a non-viral particle. [0009] In one aspect, the first plasmid is the Bv1/pCAEP plasmid, the second plasmid is the pCgpn plasmid, and the third plasmid is the pdnG1/C-REX plasmid, pdnG1/C-REX II plasmid, pdnG1/UBER-REX plasmid. [0010] The collected particles generally exhibit a viral titer of about 1.times.10.sup.7 to 1.times.10.sup.11, 1.times.10.sup.8 to 1.times.10.sup.11, 1.times.10.sup.9 to 1.times.10.sup.11, 5.times.10.sup.8 to 5.times.10.sup.10, or 1.times.10.sup.9 to 5.times.10.sup.11, at least 5.times.10.sup.8, 1.times.10.sup.9, 5.times.10.sup.9, 1.times.10.sup.10, 5.times.10.sup.10, or 1.times.10.sup.11 colony forming units per milliliter. In addition, the viral particles are generally about 10 nm to 1000 nm, 20 nm to 500 nm, 50 nm to 300 nm, 50 nm to 200 nm, or 50 nm to 150 nm in diameter. [0011] In one embodiment, the collagen binding domain includes a peptide derived from the D2 domain of von Willebrand factor. Generally, the von Willebrand factor is derived from a mammal. The peptide includes the amino acid sequence Gly-His-Val-Trp-Arg-Glu-Pro-Ser-Phe Met-Ala-Lys-Ser-Ala-Ala (SEQ ID NO:1), or Gly-His-Val-Gly-Trp-Arg-Glu-Pro-Ser-Phe Met- Ala-Lys-Ser-Ala-Ala (SEQ ID NO:8). [0012] In another embodiment, the peptide is contained in the gp70 portion of the 4070A amphotropic envelope protein. [0013] In another embodiment, the therapeutic polypeptide is an N-terminal deletion mutant of cyclin G1, interleukin-2 (IL-2), granulocyte macrophage-colony stimulating factor (GM-CSF), or thymidine kinase. [0014] Targeted delivery vectors disclosed herein generally contain nucleic acid sequences encoding diagnostic or therapeutic polypeptides. As described in greater detail in other portions of this specification, exemplary therapeutic proteins and polypeptides of the invention include, but are in no way limited to, those of the classes of suicidal proteins, apoptosis-inducing proteins, cytokines, interleukins, and TNF family proteins. Exemplary diagnostic proteins or peptides, include for example, a green fluorescent protein and luciferase. [0015] In another embodiment, a method of treating a subject having a neoplastic disorder is provided. The method include a first phase protocol comprising contacting a subject with a viral particle described herein, wherein the subject is contacted with i) a first viral particle dose level of about 1.times.10.sup.9 to 6.times.10.sup.9 Units/day administered to the subject 1 to about 6 days in succession; ii) a second viral particle dose level of about 7.times.10.sup.9 to about 1.times.10.sup.10 Units/day administered to the subject for 1 to about 3 days in succession and subsequent to administration of the first vector dose; and iii) a viral particle dose level of about 1.times.10.sup.10 to about 5.times.10.sup.10 Units/day administered to the subject for 1 to about 3 days in succession and subsequent to administration of the second vector dose. The method further includes a second phase protocol comprising contacting a subject with a viral particle produced as described herein, wherein the subject is contacted with a viral particle dose level of about 1.times.10.sup.9 to about 5.times.10.sup.10 Units/day administered to subject for 1 to about 15 days in succession and subsequent to the first phase protocol. The method optionally includes administering a chemotherapeutic agent to the subject prior to, contemporaneously with, or subsequent to the phase one and phase two protocols. The first viral particle dose level can be about 4.times.109 to 5.times.109 Units/day. The second viral particle dose level can be about 9.times.109 to about 1.times.10.sup.10 Units/day. The third viral particle dose level can be about 1.times.10.sup.10 to about 2.times.10.sup.10 Units/day. Generally the viral particles accumulate in the subject in areas of exposed collagen. Such areas of exposed collagen include neoplastic lesions, areas of active angiogenesis, neoplastic lesions, areas of vascular injury, surgical sites, inflammatory sites and areas of tissue destruction. [0016] Targeted delivery vectors disclosed herein can be administered topically, intravenously, intra-arterially, intracolonically, intratracheally, intraperitoneally, intranasally, intravascularly, intrathecally, intracranially, intramarrowly, intrapleurally, intradermally, subcutaneously, intramuscularly, intraocularly, intraosseously and/or intrasynovially. [0017] In another embodiment, a plasmid including a multiple cloning site functionally-linked to a promoter, wherein the promoter supports expression of a heterologous nucleic acid sequence; 5' and 3' long terminal repeat sequences; a .PSI. retroviral packaging sequence; a CMV promoter positioned upstream of the 5' LTR; a nucleic acid sequence operably linked to a promoter, wherein the sequence encodes a polypeptide that confers drug resistance on a producer cell containing the plasmid; and an SV40 origin of replication. Exemplary plasmids include pC-REX II, pC-REX and pUBER-REX. Additional derivatives of the exemplary include those that contain a heterologous nucleic acid sequence encoding a therapeutic or diagnostic polypeptide. [0018] In other embodiments, a kit for the production of targeted delivery vectors is provided. The kit generally includes containers containing plasmids disclosed herein for the production of, for example, viral particle. Such kits can further include a producer cell suitable for transfecting with the plasmids, and instructions for transiently transfecting the producer cell with the plasmids. The instructions can further include methods for culturing the transfected producer cell under conditions that allow targeted delivery vectors to be produced. For example, a kit for the production of targeted viral particles can include containers containing the Bv1/pCAEP plasmid, the pCgpn plasmid, and the pdnG1/C-REX plasmid, the pdnG1/C-REX II plasmid, or the pdnG1/UBER-REX plasmid. Such a kit can further include 293T cells and instructions for transiently transfecting cells with the plasmids and culturing the transfected cell under conditions that allow targeted viral particles to be produced. [0019] In another embodiment, a kit for treating a neoplastic disorder is provided. The kit includes a container containing a viral particle produced by a method described herein in a pharmaceutically acceptable carrier and instructions for adrninistering the viral particle to a subject. The administration can be according to the exemplary treatment protocol provided in Table 1. [0020] In another embodiment, a method for conducting a gene therapy business is provided. The method includes generating targeted delivery vectors and establishing a bank of vectors by harvesting and suspending the vector particles in a solution of suitable medium and storing the suspension. The method further includes providing the particles, and instructions for use of the particles, to a physician or health care provider for administration to a subject (patient) in need thereof. Such instructions for use of the vector can include the exemplary treatment regimen provided in Table 1. The method optionally includes billing the patient or the patient's insurance provider. 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