DNA composition and uses thereof

Abstract

A plasmid DNA that encodes one or more antigenic genes operably linked to a promoter and a truncated retroviral 3′ LTR exhibits both enhanced safety and acceptable efficiency of expression of antigenic proteins.

1  and  2 . (canceled) 
     
     
         3 . A DNA construct comprising
 a) a promoter capable of directing transcription in Langerhans or dendritic cells;   b) a multiplicity of regulatory and structural genes and regulatory sequences of a retrovirus arranged in the same manner as in the wild-type virus, operably linked to said promoter, wherein one or more gene(s) may comprise deletion(s) or truncation(s); and   d) a truncated retroviral 3′ LTR preferably comprising at least a part of each protein coding sequence originally comprised in said LTR, said part encoding an immunogenic portion of the said protein, wherein the said truncation disables reverse transcription of the retroviral sequences comprised in the DNA construct and the promoter function of said LTR,   wherein said construct, when complexed with polyethylenimine-mannose, being capable of eliciting an antiretroviral immune response in mammals upon endocytosis by Langerhans or dendritic cells.   
     
     
         4 . The DNA construct of  claim 3  wherein said promoter is a promoter derived from a 5′ retroviral LTR, or a functional part of said LTR. 
     
     
         5 . The DNA construct of  claim 3  or  4  wherein said promoter is derived from a 5′ HIV-LTR. 
     
     
         6 . The DNA construct of any one of  claims 3 - 5  wherein said DNA construct comprises all regulatory sequences in functional form and at least a portion of every structural and regulatory genes of said retrovirus, said structural and regulatory genes encoding the entirety or an immunogenic portion of each and every protein of said retrovirus. 
     
     
         7 . The DNA construct of any one of  claims 3 - 6  wherein said DNA construct comprises a mutant gene expressing a dominant negative mutant viral protein. 
     
     
         8 . The DNA construct of any one of  claims 3 - 7  wherein said retrovirus is HIV and said 3′ LTR is a 3′ HIV-LTR. 
     
     
         9 . The DNA construct of  claim 8  wherein said 3′ HIV-LTR comprises a deletion involving the R region and/or the U5 region and/or the TATA signal and the transcriptional factor binding elements NF-kappaB and SP1. 
     
     
         10 . The DNA construct of  claim 8  or  9  wherein said truncated 3′ HIV-LTR is the one depicted at  FIG. 2  the sequence of which is comprised in SEQ. ID. NO: 2 (from nt. position 9083 till nt position 9243). 
     
     
         11 . The DNA construct of any one of  claims 3 - 10  wherein said DNA construct comprises a mutant integrase gene which expresses a protein lacking integrase activity. 
     
     
         12 . The DNA construct of  claim 11  wherein said DNA construct comprises a mutant integrase gene which expresses a protein capable of eliciting an integrase specific immune response. 
     
     
         13 . The DNA construct of  claim 11  or  12  wherein said mutant integrase gene comprises  6  stop codon mutations at nt. 4657-4659. ni. 4663-4665, nt. 4679-4681, nt 4682-4684, nt. 4691-4693 and nt. 4703-4705 of SEQ. ID. NO: 2, respectively, and one 7 base pair deletion from nt. 4669 to nt. 4675 of SEQ. ID. NO: 2. 
     
     
         14 . The DNA construct of any one of  claims 3 - 13  wherein said DNA construct comprises a mutant gag gene which expresses a protein comprising a disabled protease cleavage site though being capable of being incorporated into the viral particle and eliciting a gag-specific immune response. 
     
     
         15 . The DNA construct of  claim 14  wherein said mutant gag gene comprises the deletion depicted at  FIG. 5  eliminating nucleotides 1097-1267 of SEQ. ID. NO: 1. 
     
     
         16 . The DNA construct of any one of  claims 3 - 13  wherein said DNA construct comprises the promoter of  claim 5 , the truncated LTR of  claim 10 , the mutant integrase gene of  claim 13 , a mutant nef gene having the sequence of nt. 8794-9246 of SEQ. ID. NO: 2 and wild-type HIV genes of tat, rev, vpu, vpr. vif, gag, reverse transcriptase, protease and envelope, all having the sequences as shown within SEQ. ID. NO: 1, respectively. 
     
     
         17 . The DNA construct of any one of  claims 3 - 15  wherein said DNA construct comprises the promoter of  claim 5 , the truncated LTR of  claim 1 , the mutant integrase gene of  claim 13 , the mutant gag gene of  claim 15 , a mutant nef gene having the sequence of nt. 8794-9246 of SEQ. ID. NO: 2 and wild-type HIV genes of tat, rev, vpu, vpr, vif, reverse transcriptase protease and envelope, all having the sequences as shown within SEQ. ID. NO: 1, respectively. 
     
     
         18 . A method of using the DNA construct of any one of  claims 3 - 17  for prevention or treatment of retroviral infection comprising administering an effective amount of the construct using a physiologically acceptable formulation via a route selected from the group comprising intramuscular, subcutaneous, intradermal, aerosolic, rectal, vaginal, oral or topical. 
     
     
         19 . The method of  claim 18 , wherein the route is topical and wherein the stratum corneum is perforated prior to topical administration. 
     
     
         20 . The method of  claim 19 , wherein the stratum corneum of a selected area of the skin is perforated in a degree sufficient to access Langerhans cells and a therapeutically effective amount of DNA formulation is applied on the said selected area of the skin. 
     
     
         21 . The method of  claim 20 , wherein the said perforation is conducted with a method selected from the group of shaving, clipping, stripping, exfoliating, rubbing, scarifying and scratching the skin.