Retroviral vector for the administration and expression of replicon rna expressing heterologous nucleic acids

Abstract

The present disclosure relates generally to gene delivery using a chimeric, retroviral-RNA replicon vector particle for increased expression of transgenes in a host cell. In particular, the chimeric vectors described herein can be used in any of a variety of settings including gene therapy and vaccine settings.

1 .- 3 . (canceled) 
     
     
         4 . A chimeric retroviral-RNA replicon vector particle, comprising:
 a) an envelope glycoprotein;   b) a retroviral gag polyprotein comprising matrix, capsid and nucleocapsid proteins;   c) a retroviral protease; and   d) an alphaviral replicon sequence comprising in a 5′ to 3′ direction: i) a polynucleotide sequence comprising an alphavirus 5′ untranslated region (5′UTR); ii) a polynucleotide sequence encoding a full-length replicase polyprotein (REP) wherein the polynucleotide sequence, following an opal stop codon in nsP3, which polynucleotide sequence encodes a proteolytic cleavage recognition site, is duplicated and wherein either a retroviral packaging signal and an IRES or a retroviral packaging signal modified to contain no stop codons, is inserted between the duplicated sequence encoding the proteolytic cleavage recognition site; and iii) a heterologous nucleic acid sequence of interest (SOI), wherein the SOI does not encode a), b), or c).   
     
     
         5 . The chimeric retroviral-RNA replicon vector particle of  claim 4 , wherein the proteolytic cleavage recognition site comprises the amino acid sequence set forth in SEQ ID NO:4. 
     
     
         6 . The chimeric retroviral-RNA replicon vector particle of  claim 4 , wherein the vector genome further comprises a hepatitis D ribozyme sequence. 
     
     
         7 . The chimeric retroviral-RNA replicon vector particle of  claim 4 , wherein the retroviral packaging sequence is a lentiviral packaging sequence. 
     
     
         8 . (canceled) 
     
     
         9 . The chimeric retroviral-RNA replicon vector particle of  claim 4 , wherein the alphaviral replicon sequence is derived from an alphavirus selected from the group consisting of Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, Middelburg virus, Ndumu virus, Semliki Forest virus, Bebaru virus, Chikungunya virus, Mayaro virus, Ross River virus, Getah virus, Aura virus, Babanki virus, Sindbis virus, Ockelbo virus, and Whataroa virus. 
     
     
         10 . The chimeric retroviral-RNA replicon vector particle of  claim 4 , wherein the envelope glycoprotein comprises a VSVg, an alphavirus E2 glycoprotein, a retroviral envelope glycoprotein, or a targeting antibody. 
     
     
         11 .- 16 . (canceled) 
     
     
         17 . The chimeric retroviral-RNA replicon vector particle of  claim 4 , wherein the SOI encodes a tumor associated antigen, a viral antigen, a bacterial antigen, or a fungal antigen. 
     
     
         18 . (canceled) 
     
     
         19 . The chimeric retroviral-RNA replicon vector particle of  claim 4 , wherein the retroviral vector genome is a lentiviral vector genome. 
     
     
         20 . A pharmaceutical composition comprising the chimeric retroviral RNA replicon vector particle of  claim 4 . 
     
     
         21 . A method of inducing an immune response in a subject, comprising administering to the subject a pharmaceutical composition comprising the chimeric retroviral-RNA replicon vector particle of  claim 17 . 
     
     
         22 . (canceled) 
     
     
         23 . A method of treating cancer in a subject, comprising administering to the subject the pharmaceutical composition of  claim 20 , wherein the SOI encodes one or more tumor associated antigens and optionally one or more immune checkpoint inhibitors or one or more cytokines, or a combination thereof. 
     
     
         24 . The method of  claim 23 , further comprising administering an additional therapeutic agent. 
     
     
         25 . The method of  claim 24 , wherein the additional therapeutic agent is selected from the group consisting of a cytokine, an immune checkpoint inhibitor, a TLR agonist, a chemotherapeutic agent, and radiation. 
     
     
         26 . A method of treating an infectious disease in a subject, comprising administering to the subject a pharmaceutical composition of  claim 20  wherein the SOI encodes an antigen associated with the infectious disease. 
     
     
         27 .- 29 . (canceled) 
     
     
         30 . A packaging system for producing a chimeric retroviral-RNA replicon vector particle, wherein the particle is reverse transcriptase independent, comprising a packaging cell transfected or otherwise modified to contain:
 a) a first nucleic acid molecule encoding an envelope;   b) a second nucleic acid molecule encoding gag and pol proteins, wherein the nucleic acid molecule optionally encodes a nonfunctional reverse transcriptase protein;   c) a third nucleic acid molecule comprising an alphaviral replicon sequence comprising in a 5′ to 3′ direction: i) a polynucleotide sequence comprising an alphavirus 5′ untranslated region (5′UTR); ii) a polynucleotide sequence encoding a full-length replicase polyprotein (REP) wherein the polynucleotide sequence, following an opal stop codon in nsP3, which polynucleotide sequence encodes a proteolytic cleavage recognition site, is duplicated and wherein either a retroviral packaging signal and an IRES or a retroviral packaging signal modified to contain no stop codons, is inserted between the duplicated sequence encoding the proteolytic cleavage recognition site; and iii) a heterologous nucleic acid sequence of interest (SOI).   
     
     
         31 . The packaging system of  claim 30 , wherein the proteolytic cleavage recognition site comprises the amino acid sequence set forth in SEQ ID NO:4. 
     
     
         32 . The packaging system of  claim 30 , wherein the envelope glycoprotein comprises a VSVg or an alphavirus E2 glycoprotein. 
     
     
         33 . The packaging system of  claim 30 , wherein the retroviral vector genome comprises a 3′ polypurine tract (PPT) which has been deleted or otherwise mutated to be nonfunctional. 
     
     
         34 . The packaging system of  claim 30 , wherein the pol protein comprises a nonfunctional integrase. 
     
     
         35 . (canceled) 
     
     
         36 . A method of producing a chimeric retroviral-RNA replicon vector particle comprising culturing the packaging cell of  claim 30 . 
     
     
         37 .- 38 . (canceled) 
     
     
         39 . A therapeutic or prophylactic vaccine comprising the chimeric retroviral-RNA replicon vector particles of  claim 4  and a pharmaceutically acceptable excipient.