Non-integrating viral delivery system and methods related thereto

A method of expressing at least one gene, gene product, shRNA, siRNA, miRNA, or other RNA of interest in a cell, the method comprising:

• contacting the cell with a non-integrating viral delivery system, wherein the system comprises:
• (a) a viral carrier, wherein the viral carrier is a lentiviral vector and contains a defective integrase gene;
• (b) a first sequence encoding:
• i. a heterologous episomal HPV16 origin of DNA replication comprising a 5′ truncated HPV16 Long Control Region (LCR),
• wherein the 5′ truncated HPV16 LCR comprises:
• a. an E1 binding site, two E2 binding sites and an SP1 binding site;
• or
• b. an E1 binding site, three E2 binding sites and an SP1 binding site;
• or
• c. at least two AP1 binding sites, an E1 binding site, three E2 binding sites and an SP1 binding site, wherein none of the AP1 binding sites are outside of the HPV16 LCR enhancer region;
• or
• ii. a heterologous episomal HPV16 origin of DNA replication comprising a 5′ truncated HPV16 LCR and at least one initiator protein specific for the heterologous episomal HPV16 origin of replication,
• wherein the 5′ truncated HPV16 LCR comprises:
• a. an E1 binding site, two E2 binding sites and an SP1 binding site;
• or
• b. an E1 binding site, three E2 binding sites and an SP1 binding site;
• or
• c. at least two AP1 binding sites, an E1 binding site, three E2 binding sites and an SP1 binding site, wherein none of the AP1 binding sites are outside of the HPV16 LCR enhancer region;
• wherein the at least one initiator protein comprises:
• E1, an E1 operative variant, E1-C, E2, an E2 operative variant,
• E1-T2A-E2, EBNA-1, or an EBNA-1 operative fragment;
• or
• iii. a heterologous episomal HPV16 origin of DNA replication comprising a 5′ truncated HPV16 LCR,
• wherein the 5′ truncated HPV16 LCR comprises:
• a. at least three AP1 binding sites, an E1 binding site, four E2 binding sites and one SP1 binding site,
• wherein one AP1 binding site is outside of the HPV16 LCR enhancer region;
• or
• iv. a heterologous episomal HPV16 origin of DNA replication comprising a 5′ truncated HPV16 LCR and at least one initiator protein specific for the heterologous episomal HPV16 origin of replication,
• wherein the 5′ truncated HPV16 LCR comprises:
• a. at least three AP1 binding sites, an E1 binding site, four E2 binding sites and one SP1 binding site,
• wherein one AP1 binding site is outside of the HPV16 LCR enhancer region;
• wherein the at least one initiator protein comprises:
• E1, an E1 operative variant, E1-C, E2, an E2 operative variant,
• E1-T2A-E2, EBNA-1, or an EBNA-1 operative fragment;
• (c) a second sequence encoding at least one gene, gene product, shRNA, siRNA, miRNA, or other RNA of interest.