Pathotropic targeted gene delivery system for cancer and other disorders

Abstract

Systems for pathotropic (disease-seeking) targeted gene delivery are provided, including viral particles with extremely high titers. In particular, the viral particles are engineered to specifically deliver therapeutic or diagnostic agents to a disease site, such as cancer metastic sites. Personalized dosing regimens are also provided to treat diseases such as cancer efficaciously with reduced adverse side effects.

1 . A method of treating a subject having a tumor or tumors containing cancer cells with therapeutic viral particles, the method comprising: 
 a) determining the dose of the therapeutic viral particles by 
 i) determining the subject's tumor burden as defined by the number of cancer cells residing in the subject's tumor, or the total number of tumor cells in the tumors;  
 ii) multiplying the tumor burden by the physiological mulitplicity of infection (pMOI) of the therapeutic viral particles; and  
 iii) dividing the resultant figure by the titer of the therapeutic viral particles to yield the volume of the therapeutic viral particles to be administered to the subject; and  
   b) administering the determined dose of the therapeutic viral particles to the subject.    
     
     
         2 . The method of  claim 1 , wherein the subject is treated with the therapeutic viral particles at the determined dose of the therapeutic viral particles per day for 1 to about 6 days in succession.  
     
     
         3 . The method of  claim 1 , wherein the subject is treated with the therapeutic viral particles at the determined dose of the therapeutic viral particles per day for 1 to 5 days in succession.  
     
     
         4 . The method of  claim 1 , wherein the subject is treated with the therapeutic viral particles at the determined dose of the therapeutic viral particles per day for on Monday, Wednesday, and Friday in succession.  
     
     
         5 . The method of  claim 2 , wherein the subject is allowed to rest 1 to 2 days, which constitutes a treatment cycle.  
     
     
         6 . The method of  claim 5 , wherein the subject is treated for 2-8 treatment cycles.  
     
     
         7 . The method of  claim 5 , wherein the subject is treated for 3-4 treatment cycles.  
     
     
         8 . The method of  claim 1 , wherein the dose of the therapeutic viral particles in a unit of milliliters is calculated based on the following general formula:  
       
         
           
             
               
                 Tumor 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 
                   Burden 
                   ( 
                   
                     number 
                     ⁢ 
                     
                         
                     
                     ⁢ 
                     of 
                     ⁢ 
                     
                         
                     
                     ⁢ 
                     cancer 
                     ⁢ 
                     
                         
                     
                     ⁢ 
                     cells 
                   
                   ) 
                 
                 × 
                 pMOI 
               
               
                 Viral 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 
                   Titer 
                   ⁡ 
                   
                     ( 
                     
                       colony 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       forming 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       
                         
                           units 
                           ⁡ 
                           
                             ( 
                             
                               C 
                               . 
                               F 
                               . 
                               U 
                               . 
                             
                             ) 
                           
                         
                         / 
                         ml 
                       
                     
                     ) 
                   
                 
               
             
           
         
       
       wherein pMOI is from 4 to 250.  
     
     
         9 . The method of  claim 8 , wherein pMOI is about 100.  
     
     
         10 . The method of claims  1 , further comprising: 
 after the subject is treated with the determined dose of the therapeutic viral particles, determining the tumor burden of the subject;    recalculating the dose of the therapeutic viral particles; and    administering the therapeutic viral particles to the subject at the recalculated dose.    
     
     
         11 . The method of  claim 1 , wherein the tumor burden is determined by the following formula: 
 (the sum of the longest diameters (cm) of target lesion or tumor)×1×10 9  cancer cells/cm.    
     
     
         12 . The method of  claim 11 , wherein the target lesion or tumor is measured by calipers, or by radiologic imaging.  
     
     
         13 . The method of  claim 12 , wherein the radiologic imaging is MRI, CT, PET, or SPECT scan.  
     
     
         14 . The method of  claim 1 , wherein the therapeutic viral particle is administered topically, intravenously, intra-arterially, intracolonically, intratracheally, intraperitoneally, intranasally, intravascularly, intrathecally, intracranially, intramarrowly, intrapleurally, intradermally, subcutaneously, intramuscularly, intraocularly, intraosseously and/or intrasynovially.  
     
     
         15 . The method of  claim 1 , wherein the therapeutic viral particle is administered to the subject via intravenously infusion.  
     
     
         16 . The method of  claim 1 , wherein the subject is a mammal.  
     
     
         17 . The method of  claim 1 , wherein the subject is a human.  
     
     
         18 . The method of  claim 1 , wherein the cancer is selected from the group consisting of breast cancer, skin cancer, bone cancer, prostate cancer, liver cancer, lung cancer, brain cancer, cancer of the larynx, gall bladder, pancreas, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma of both ulcerating and papillary type, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma, veticulum cell sarcoma, myeloma, giant cell tumor, small-cell lung tumor, gallstones, islet cell tumor, primary brain tumor, acute and chronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuronms, intestinal ganglloneuromas, hyperplastic corneal nerve tumor, marfanoid habitus tumor, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosis fungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and other sarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera, adenocarcinoma, glioblastoma multiforma, leukemias, lymphomas, malignant melanomas, and epidermoid carcinomas.  
     
     
         19 . The method of  claim 1 , wherein the cancer is pancreatic cancer, breast cancer, or colon cancer.  
     
     
         20 . The method of  claim 1 , wherein the viral particles accumulate in the subject in areas of exposed collagen.  
     
     
         21 . The method of  claim 20 , wherein the 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.  
     
     
         22 . The method of  claim 1 , wherein the therapeutic viral particles are a retroviral vector having an envelope protein modified to contain a collagen binding domain, and encodes a therapeutic agent against the cancer.  
     
     
         23 . The method of  claim 22 , wherein the retroviral vector is amphotropic.  
     
     
         24 . The method of  claim 22 , wherein the therapeutic agent is a cyclin G1 mutant.  
     
     
         25 . The method of  claim 22 , wherein the retroviral vector is produced by a method comprising: 
 a) transiently transfecting a producer cell with: 
 i) a first plasmid comprising a nucleic acid sequence encoding the 4070A amphotropic envelope protein modified to contain a collagen binding domain, wherein the nucleic acid sequence is operably linked to a promoter;  
 ii) a second plasmid comprising: 
 a nucleic acid sequence operably linked to a promoter, wherein the sequence encodes a viral gag-pol polypeptide,  
 a nucleic acid sequence operably linked to a promoter, wherein the sequence encodes a polypeptide that confers drug resistance on the producer cell,  
 an SV40 origin of replication;  
 
 iii) a third plasmid comprising: 
 a heterologous nucleic acid sequence operably linked to a promoter, wherein the sequence encodes a diagnostic or therapeutic polypeptide,  
 5′ and 3′ long terminal repeat sequences (LTRs),  
 a Ψ retroviral packaging sequence,  
 a CMV promoter 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 the producer cell,  
 an SV40 origin of replication,  
 wherein the producer cell is a human cell that expresses SV40 large T antigen;  
 
   b) culturing the producer cells of a) under conditions that allow targeted delivery vector production and release in to the supernatant of the culture;    c) isolating and introducing the supernatant in to a closed loop manifold system for collecting the viral particles; and    d) collecting the retroviral vectors.    
     
     
         26 . The method of  claim 25 , wherein the first plasmid is the Bv1/pCAEP plasmid.  
     
     
         27 . The method of  claim 25 , wherein the first plasmid is the pB-RVE plasmid.  
     
     
         28 . The method of  claim 25 , wherein the second plasmid is the pCgpn plasmid.  
     
     
         29 . The method of  claim 25 , wherein the third plasmid is derived from the G1XSvNa plasmid.  
     
     
         30 . The method of  claim 25 , wherein the third plasmid is the pdng1/C-REX plasmid.  
     
     
         31 . The method of  claim 25 , wherein the third plasmid is the pdng1/C-REX II plasmid.  
     
     
         32 . The method of  claim 25 , wherein the third plasmid is the pdng1/UBER-REX plasmid.  
     
     
         33 . The method of  claim 1 , further comprising: 
 administering to the subject a chemotherapeutic agent, a biologic agent, or radiotherapy prior to, contemporaneously with, or subsequent to the administration of the therapeutic viral particles.    
     
     
         34 . A plasmid comprising: 
 e) an optional multiple cloning site functionally-linked to a promoter, wherein the promoter supports expression of a heterologous nucleic acid sequence;    f) 5′ and 3′ long terminal repeat sequences (LTRs);    g) a Ψ retroviral packaging sequence;    h) a CMV promoter positioned upstream of the 5′ LTR sequence;    i) 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    j) an SV40 origin of replication.    
     
     
         35 . The plasmid of  claim 34 , wherein the plasmid is pC-REX.  
     
     
         36 . The plasmid of  claim 34 , wherein the plasmid is pC-REX II.  
     
     
         37 . The plasmid of  claim 34 , wherein the plasmid is pUBER-REX.  
     
     
         38 . The plasmid of  claim 34  further comprising a heterologous nucleic acid sequence encoding a therapeutic or diagnostic polypeptide.  
     
     
         39 . The plasmid of  claim 38 , wherein the therapeutic polypeptide is a mutant cyclin G1.  
     
     
         40 . A method for producing a targeted viral vector, the method comprising: 
 (a) transiently transfecting a producer cell with: 
 i) a first plasmid comprising a nucleic acid sequence encoding the 4070A amphotropic envelope protein modified to contain a collagen binding domain, wherein the nucleic acid sequence is operably linked to a promoter;  
 ii) a second plasmid comprising: 
 a nucleic acid sequence operably linked to a promoter, wherein the sequence encodes a viral gag-pol polypeptide,  
 a nucleic acid sequence operably linked to a promoter, wherein the sequence encodes a polypeptide that confers drug resistance on the producer cell,  
 an SV40 origin of replication;  
 
 iii) a third plasmid comprising: 
 a heterologous nucleic acid sequence operably linked to a promoter, wherein the sequence encodes a diagnostic or therapeutic polypeptide,  
 5′ and 3′ long terminal repeat sequences (LTRs),  
 a Ψ retroviral packaging sequence,  
 a CMV promoter 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 the producer cell,  
 an SV40 origin of replication,  
 wherein the producer cell is a human cell that expresses SV40 large T antigen;  
 
   (b) culturing the producer cells of a) under conditions that allow targeted delivery vector production and release in to the supernatant of the culture;    (c) isolating and introducing the supernatant in to a closed loop manifold system for collecting the viral particles; and    (d) collecting the targeted delivery vectors.    
     
     
         41 . The method of  claim 40 , wherein the collected viral particles exhibit a viral titer of about 1×10 7  to 1×10 11  colony forming units per milliliter.  
     
     
         42 . The method of  claim 40 , wherein the collected viral particles exhibit a viral titer of at least 1×10 8  colony forming units per milliliter.  
     
     
         43 . The method of  claim 40 , wherein the collected viral particles exhibit a viral titer of at least 1×10 9  colony forming units per milliliter.  
     
     
         44 . The method of  claim 40 , wherein the collected viral particles exhibit a viral titer of 1×10 8 -1×10 10  colony forming units per milliliter.  
     
     
         45 . The method of  claim 40 , wherein the collected viral particles exhibit a viral titer of 1×10 9 -1×10 10  colony forming units per milliliter.  
     
     
         46 . The method of  claim 40 , wherein the viral particles are 50 nm to 150 nm in diameter.  
     
     
         47 . The method of  claim 40 , wherein the collagen binding domain comprises a peptide derived from the D2 domain of von Willebrand factor.  
     
     
         48 . The method of  claim 47 , wherein the von Willebrand factor is bovine von Willebrand factor.  
     
     
         49 . The method of  claim 47 , wherein the peptide comprises the amino acid sequence Gly-His-Val-Trp-Arg-Glu-Pro-Ser-Phe Met-Ala-Lys-Ser-Ala-Ala (SEQ ID NO:1).  
     
     
         50 . The method of  claim 47 , wherein the peptide comprises the amino acid sequence Gly-His-Val-Gly-Trp-Arg-Glu-Pro-Ser-Phe-Met-Ala-Lys-Ser-Ala-Ala (SEQ ID NO:8).  
     
     
         51 . The method of  claim 40 , wherein the peptide is contained in the gp70 portion of the 4070A amphotropic envelope protein.  
     
     
         52 . The method of  claim 40 , wherein the therapeutic polypeptide is an N-terminal deletion mutant of cyclin G1.  
     
     
         53 . The method of  claim 52 , wherein the N-terminal deletion mutant of cyclin G1 comprises from about amino acid 41 to 249 of human cyclin G1.  
     
     
         54 . The method of  claim 40 , wherein the therapeutic polypeptide is interleukin-2 (IL-2).  
     
     
         55 . The method of  claim 40 , wherein the therapeutic polypeptide is granulocyte macrophage-colony stimulating factor (GM-CSF).  
     
     
         56 . The method of  claim 40 , wherein the therapeutic polypeptide is thymidine kinase.  
     
     
         57 . The method of  claim 40 , wherein the diagnostic polypeptide is selected from the group consisting of green fluorescent proteins, luciferase, radioisotopes, or combinations thereof.  
     
     
         58 . The method of  claim 40 , wherein the first plasmid is the Bv1/pCAEP plasmid.  
     
     
         59 . The method of  claim 40 , wherein the first plasmid is the pB-RVE plasmid  
     
     
         60 . The method of  claim 40 , wherein the second plasmid is the pCgpn plasmid.  
     
     
         61 . The method of  claim 40 , wherein the third plasmid is derived from the G1XSvNa plasmid.  
     
     
         62 . The method of  claim 40 , wherein the third plasmid is the pdnG1/C-REX plasmid.  
     
     
         63 . The method of  claim 40 , wherein the third plasmid is the pdnG1/C-REX II plasmid.  
     
     
         64 . The method of  claim 40 , wherein the third plasmid is the pdnG1/UBER-REX plasmid.  
     
     
         65 . The method of  claim 40 , wherein the producer cell is a 293T cell.  
     
     
         66 . A kit for the production of targeted viral particles, the kit comprising: 
 (a) a container containing a first plasmid comprising a nucleic acid sequence encoding the 4070A amphotropic envelope protein modified to contain a collagen binding domain, wherein the nucleic acid sequence is operably linked to a promoter;    (b) a container containing a second plasmid comprising:    a nucleic acid sequence operably linked to a promoter, wherein the sequence encodes a viral gag-pol polypeptide, a nucleic acid sequence operably linked to a promoter, wherein the sequence encodes a polypeptide that confers drug resistance on the producer cell, 
 an SV40 origin of replication;  
   (c) a container containing a third plasmid comprising:    a heterologous nucleic acid sequence operably linked to a promoter, wherein the sequence encodes a diagnostic or therapeutic polypeptide, 
 5′ and 3′ long terminal repeat sequences (LTRs),  
 a Ψ retroviral packaging sequence,  
 a CMV promoter 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 the producer cell,  
 an SV40 origin of replication, and  
   (d) a container containing a producer cell that expresses SV40 large T antigen;    
     
     
         67 . The kit of  claim 66 , wherein the first plasmid is the Bv1/pCAEP plasmid.  
     
     
         68 . The kit of  claim 66 , wherein the first plasmid is the pB-RVE plasmid.  
     
     
         69 . The kit of  claim 66 , wherein the second plasmid is the pCgpn plasmid.  
     
     
         70 . The kit of  claim 66 , wherein the third plasmid is the pdnG1/C-REX plasmid.  
     
     
         71 . The kit of  claim 66 , wherein the third plasmid is the pdng1/C-REX II plasmid.  
     
     
         72 . The kit of  claim 66 , wherein the third plasmid is the pdng1/UBER-REX plasmid.  
     
     
         73 . The kit of  claim 66 , wherein the producer cell is a 293T cell.  
     
     
         74 . The kit of  claim 66 , wherein the first plasmid is Bv1/pCAEP plasmid; the second plasmid a pCgpn plasmid; and the third plasmid a pdnG1/C-REX plasmid or a pdnG1/C-REX II plasmid.  
     
     
         75 . The kit of  claim 66 , wherein the first plasmid is a pB-RVE plasmid; the second plasmid a pCgpn plasmid; and the third plasmid a pdnG 1/UBER-REX plasmid;  
     
     
         76 . The kit of  claim 66 , further comprising: instructions for transiently transfecting the producer cell of d) with the plasmids of a), b) and c) and culturing the transfected producer cell under conditions that allow targeted viral particles to be produced.  
     
     
         77 . A kit for treating a neoplastic disorder, the kit comprising: 
 k) a container containing a targeted delivery vector produced by the method of  claim 1  in a pharmaceutically acceptable carrier; and    l) instructions for administering the vector of a) to a subject according to the method of  claim 40.