Lentiviral vectors pseudotyped with a sindbis virus envelope glycoprotein

Abstract

Lentiviral vector particles comprising a Sindbis virus E2 glycoprotein variant and a lentiviral vector genome comprising a sequence of interest are provided. A lentiviral vector particle comprising: (a) an envelope comprising a Sindbis virus E2 glycoprotein variant; and (b) a lentiviral vector genome comprising a sequence of interest; wherein the E2 glycoprotein variant facilitates infection of dendritic cells by the lentiviral vector particle, and wherein the E2 glycoprotein variant has reduced binding to heparan sulfate compared to a reference sequence (HR strain).

1 . A lentiviral vector particle comprising:
 (a) an envelope comprising a Sindbis virus E2 glycoprotein having at least one amino acid change compared to SEQ ID No. 1, wherein residue 160 is either absent or an amino acid other than glutamic acid; wherein E2 glycoprotein is not part of a fusion protein with Sindbis virus E3 protein; and   (b) a lentiviral vector genome comprising a sequence of interest,   wherein the E2 glycoprotein facilitates infection of dendritic cells by the lentiviral vector particle.   
     
     
         2 . The lentiviral vector particle of  claim 1 , wherein the E2 glycoprotein binds to DC-SIGN. 
     
     
         3 . The lentiviral vector particle of  claim 1 , wherein residue 160 of E2 is changed to Gly. 
     
     
         4 . The lentiviral vector particle of  claim 1 , wherein residue 159 of E2 is changed to Glu 
     
     
         5 . The lentiviral vector particle of  claim 1 , wherein at least one other amino acid alteration reduces net positive charge of E2 glycoprotein. 
     
     
         6 . The lentiviral vector particle of  claim 5 , wherein residues 70 or 76 or both of E2 are changed to Glu or Asp. 
     
     
         7 . The lentiviral vector particle of  claim 1 , wherein the E2 glycoprotein has a sequence set forth in SEQ ID NOs. 3, 4, or 5. 
     
     
         8 . The lentiviral vector particle of  claim 1 , wherein the sequence of interest encodes a tumor-specific antigen or a virus-derived antigen. 
     
     
         9 . The lentiviral vector particle of  claim 8 , wherein the tumor-specific antigen is selected from the group consisting of carbonic anhydrase IX, NKX 3.1, her2/neu, PSA and p53. 
     
     
         10 . The lentiviral vector particle of  claim 8 , wherein the virus-derived antigen is selected from the group consisting of GP120 of HIV-1, gD of HSV-2, Hepatitis B surface antigen and influenza HA. 
     
     
         11 . The lentiviral vector particle of  claim 1 , wherein the lentiviral vector genome is non-integrating. 
     
     
         12 . The lentiviral vector particle of  claim 11 , wherein the genome is deleted for PPT or is mutated or deleted for an att site. 
     
     
         13 . A lentiviral vector packaging system for producing a pseudotyped lentiviral vector particle, comprising:
 (i) a first nucleic acid molecule encoding a Sindbis virus E2 glycoprotein having at least one amino acid change compared to SEQ ID No. 1, wherein residue 160 is either absent or an amino acid other than glutamic acid; wherein E2 glycoprotein is not part of a fusion protein with Sindbis virus E3 protein,   (ii) a second nucleic acid molecule comprising a lentiviral vector genome comprising a sequence of interest;   (iii) a third nucleic acid molecule encoding gag and pol proteins; and   (iv) a fourth nucleic acid molecule encoding rev.   
     
     
         14 . The packaging system of  claim 13 , wherein the pol protein has a non-functional integrase. 
     
     
         15 . The packaging system of  claim 13 , wherein the lentiviral vector genome is non-integrating. 
     
     
         16 . The packaging system of  claim 13 , wherein the lentiviral vector particles are produced to a titer of at least 10 5  IU/mL. 
     
     
         17 . A cell comprising the nucleic acid molecules of  claim 13 . 
     
     
         18 . An isolated nucleic acid molecule comprising a sequence encoding a Sindbis virus E2 glycoprotein having at least one amino acid change compared to SEQ ID No. 1, wherein residue 160 is either absent or is an amino acid other glutamic acid; wherein residue 1 of the E2 glycoprotein is Ser. 
     
     
         19 . The isolated nucleic acid molecule of  claim 18 , further comprising a nucleic acid sequence encoding Sindbis virus E3 that is operatively linked to E2 coding sequence, wherein E2 and E3 are produced as a polyprotein and wherein there is a cleavable sequence between E3 and E2. 
     
     
         20 . An expression vector comprising the nucleic acid molecule of  claim 18 . 
     
     
         21 . A host cell comprising the expression vector of  claim 20 . 
     
     
         22 . A method of delivering a sequence of interest to dendritic cells in a subject, comprising: administering to a subject a lentiviral vector particle comprising:
 (a) an envelope comprising a Sindbis virus E2 glycoprotein having at least one amino acid change compared to SEQ ID No. 1, wherein residue 160 is either absent or is an amino acid other than glutamic acid; wherein E2 glycoprotein is not part of a fusion protein with Sindbis virus E3 protein; and   (b) a lentiviral vector genome comprising a sequence of interest,   wherein the E2 glycoprotein facilitates infection of dendritic cells by the lentiviral vector particle.   
     
     
         23 . A method of inducing an immune response to an antigen in a subject, comprising immunizing the subject with a lentiviral vector particle comprising:
 (a) an envelope comprising a Sindbis virus E2 glycoprotein having at least one amino acid change compared to SEQ ID No. 1, wherein residue 160 is either absent or an amino acid other than glutamic acid; wherein E2 glycoprotein is not part of a fusion protein with Sindbis virus E3 protein; and   (b) a lentiviral vector genome comprising sequence encoding an antigen;   wherein the E2 glycoprotein facilitates infection of dendritic cells by the lentiviral vector particle.   
     
     
         24 . A therapeutic or prophylactic vaccine comprising:
 a pharmaceutically acceptable excipient and lentiviral vector particles comprising:   (a) an envelope comprising a Sindbis virus E2 glycoprotein having at least one amino acid change compared to SEQ ID No. 1, wherein residue 160 is either absent or an amino acid other than glutamic acid; wherein E2 glycoprotein is not part of a fusion protein with Sindbis virus E3 protein; and   (b) a lentiviral vector genome comprising sequence encoding an antigen;   wherein the E2 glycoprotein facilitates infection of dendritic cells by the lentiviral vector particle.   
     
     
         25 . A lentiviral vector particle comprising:
 (a) an envelope comprising a Sindbis virus E2 glycoprotein that preferentially targets dendritic cells; wherein E2 is not part of a fusion protein with Sindbis virus E3 protein; and   (b) a lentiviral vector genome comprising a sequence of interest;   wherein the particle can be produced in unprocessed supernatant of packaging cell lines to at least 1×10 5  IU/mL.   
     
     
         26 . The particle of  claim 25 , wherein residue 160 is a non-acidic amino acid or is deleted. 
     
     
         27 . The particle of  claim 25 , wherein the lentiviral vector genome is non-integrating.