Using cytosine deaminases to diminish retroelement transfer from pigs to humans

Inventors

HARRIS, REUBEN S. • JONSSON, STEFAN R. • Fahrenkrug, Scott C. • LARUE, REBECCA ST. CLAIRE

Assignees

Recombinetics Inc

Abstract

Transgenic pigs that express one or more non-porcine cytosine deaminases are described as well as methods of making and using such pigs.

Core Innovation

The disclosure relates to transgenic pigs and porcine cells that express a non-porcine cytosine deaminase polypeptide, including non-porcine APOBEC3 cytosine deaminases such as human APOBEC3G and APOBEC3F, and other APOBEC family members. The nucleated cells comprise a nucleic acid construct with a transcriptional unit that includes a regulatory region operably linked to a nucleic acid sequence encoding the non-porcine APOBEC3 cytosine deaminase polypeptide.

Expression of these non-porcine cytosine deaminases is used to affect retroelement transfer involving porcine endogenous retroviruses (PERVs). The problem addressed is the reduction of transmission of porcine endogenous retroviruses (PERVs) from porcine sources to human cells.

The disclosure describes that cytosine deamination-driven mutator phenotypes and retrotransposition inhibition can be mediated through cytosine deamination mechanisms, including C→U deamination and G→A hypermutation. It further describes that APOBEC3 family members can inhibit retrotransposition via a cDNA cytosine deamination mechanism and that activity can be dependent on catalytic domains.

The disclosure provides supporting evidence that human APOBEC3G expressed in pig PK-15 cells decreases long-term PERV transfer to human 293T cells, including measurements by RT activity and PERV pol gene PCR. The nucleic acid construct embodiments include porcine regulatory regions, optional constitutive or tissue-specific promoters, and transcriptional-unit flanking features including insulator elements and inverted repeats of a transposon, with optional transposon/transposase components.

Claims Coverage

The partial content includes one independent claim defining a transgenic pig with a nucleic acid construct encoding a non-porcine APOBEC3 cytosine deaminase. The dependent claims refine the specific APOBEC3 enzyme, the regulatory region type, additional genetic architecture features, and a functional outcome tied to decreased PERV transmission during co-culture with human cells.

Overall, the claim coverage centers on transgenic pigs whose nucleated cells carry a transcriptional unit driven by a regulatory region to express a non-porcine APOBEC3 cytosine deaminase polypeptide, with dependent refinements specifying particular APOBEC3 members, promoter type, genetic flanking features, and a functional outcome of decreased PERV transmission during co-culture with human cells.

Stated Advantages

Documented Applications