EXOGENOUS GENE EXPRESSION IN RECOMBINANT ADENOVIRUS FOR MINIMAL IMPACT ON VIRAL KINETICS
Recombinant adenovirus genomes that include an exogenous open reading frame (ORF) and a self-cleaving peptide coding sequence are described. Optimal placement of the exogenous genes for minimal impact on viral kinetics is further disclosed. Therapeutic applications of the recombinant adenoviruses are also described.
1 . A recombinant adenovirus genome, comprising a heterologous open reading frame (ORF) and a self-cleaving peptide coding sequence, both operably linked to and in the same reading frame as an endogenous adenovirus ORF, wherein the self-cleaving peptide coding sequence is located between the heterologous ORF and the endogenous ORF, and wherein:
the endogenous ORF is E1B-55k and the heterologous ORF is 3′ of E1B-55k;
the endogenous ORF is DNA polymerase and the heterologous ORF is 5′ of DNA polymerase;
the endogenous ORF is DNA-binding protein (DBP) and the heterologous ORF is 3′ of DBP;
the endogenous ORF is adenovirus death protein (ADP) and the heterologous ORF is 5′ of ADP;
the endogenous ORF is E3-14.7k and the heterologous ORF is 3′ of E3-14.7k;
the endogenous ORF is E4-ORF2 and the heterologous ORF is 5′ of E4-ORF2; or
the endogenous ORF is fiber and the heterologous ORF is 3′ of fiber,
wherein the heterologous ORF encodes a therapeutic protein.
2 . The recombinant adenovirus genome of claim 1 , wherein the therapeutic protein comprises an immunomodulator.
3 . The recombinant adenovirus genome of claim 1 , wherein the self-cleaving peptide is a 2A peptide or variant thereof.
4 . The recombinant adenovirus genome of claim 3 , wherein the 2A peptide comprises a porcine teschovirus-1 (PTV1) 2A (P2A) peptide, a foot and mouth disease virus (FMDV) 2A (F2A) peptide, an equine rhinitis A virus (ERAV) 2A (E2A) peptide or a Thosea asigna virus (TaV) 2A (T2A) peptide, or a variant thereof.
5 . The recombinant adenovirus genome of claim 4 , wherein the amino acid sequence of the self-cleaving peptide is at least 80%, at least 85%, at least 90% or at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 14-21.
6 . The recombinant adenovirus genome of claim 4 , wherein the self-cleaving peptide comprises the amino acid sequence of any one of SEQ ID NOs: 14-21.
7 . A composition comprising the recombinant adenovirus genome of claim 1 and a pharmaceutically acceptable carrier.
8 . A recombinant adenovirus comprising the recombinant adenovirus genome of claim 1 .
9 . A composition comprising the recombinant adenovirus of claim 8 and a pharmaceutically acceptable carrier.
10 . A method of delivering a therapeutic protein to a subject, comprising administering to the subject the recombinant adenovirus genome of claim 1 .
11 . A method of inhibiting tumor cell viability and/or tumor cell growth, comprising contacting the tumor cell with the recombinant adenovirus genome of claim 1 .
12 . The method of claim 11 , wherein the method is an in vitro method.
13 . The method of claim 11 , wherein the method is an in vivo method and contacting the tumor cell comprises administering the recombinant adenovirus genome, recombinant adenovirus, or composition to a subject with a tumor.
14 . A method of inhibiting tumor progression or reducing tumor volume in a subject, comprising administering to the subject a therapeutically effective amount of the recombinant adenovirus genome of claim 1 , thereby inhibiting tumor progression or reducing tumor volume in the subject.
15 . A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the recombinant adenovirus genome of claim 1 , thereby treating cancer in the subject.
16 . The method of claim 13 , further comprising administering an additional therapeutic agent to the subject.
17 . A kit comprising:
(i) the recombinant adenovirus genome of claim 1 ; and
(ii) one or more additional therapeutic agents and/or one or more diagnostic agents.
18 . The kit of claim 17 , wherein the one or more additional therapeutic agents comprise a chemotherapeutic, biologic, or combinations thereof.
19 . The kit of claim 17 , wherein the one or more diagnostic agents comprise one or more antibodies specific for a tumor marker.
20 . The kit of claim 17 , wherein the one or more diagnostic agents comprise one or more nucleic acid molecules specific for a tumor marker.