These engineered recombinase enzymes have the potential to provide protection from HIV-1 in a manner suitable for delivery in developing nations.
In response to foreign antigens, the body’s immune system concentrates specific white blood cells at the site of infection.
Furthermore, recombinase enzymes are unlikely to suffer the same disadvantages as zinc finger nuclease enzymes because recombinase-mediated DNA excision does not rely on the generation of DNA damage as a means to effect a application, which is a necessity for conditions in the developing world.
Site-specific recombinase enzymes are ideal for genome modification applications due to their ability to recognize precise DNA sequences and remove, replace, or invert the flanked sequence (Gordley 2007).
Although rare in Africans and Asians, up to 20% of Caucasians are heterozygous Δ32 allele carriers.
In addition to having fewer functional CCR5 receptors due to Δ32 heterozygosity, the truncated protein may reduce wild-type CCR5 and CXCR4 cell-surface expression by forming heterodimers with them, which are retained in the endoplasmic reticulum (de Silva and Stumpf 2004; Agrawal -∆32 allele experience a delayed onset of AIDS by two to three years if infected with HIV-1.
After determining efficient recombinase variants individually, I will test the evolved enzymes for recombinase-mediated excision on and subsequently in human cells. Individually, the engineered enzymes show both activity and substrate specificity with regards to their target sequences on , selected active mutants, and performed recombinase activity assays side-by-side with starting pool samples. Liu *Harvard University, [email protected] designed and created enzymes consisting of the catalytic domain of Hin-H107Y, a hyperactive mutant DNA invertase, fused via a flexible linker sequence to engineered zinc finger DNA-binding domains.Zinc finger nucleases have been shown to induce toxicity as a result of DNA cleavage at non-target sites, resulting in cell death and apoptosis, and so would be ill-suited for Figure 2. The sequence of interest (arrow) between recombinase enzyme recognition sites (boxes) is excised, creating a freed loop of DNA (Akopian et al.2003).which results in a similarly-truncated receptor. If successful, this project will further efforts to confer resistance to HIV-1 infection, or to possibly treat infected individuals by generating a reserve of resistant cells.Furthermore, the 1% of Caucasians who are homozygous for the allele are nearly fully resistant to infection by the HIV-1 virus (Y. These genome-editing enzymes introduce a double-stranded DNA break which is corrected via homologous recombination with a provided ∆32 template strand.As a therapy, this method requires adoptive transfer of 2008), which is not a feasible treatment for use in developing nations.These domains are physically separated and connected by a flexible linker.Because sequence recognition and catalysis functions of a recombinase can be specified by unrelated protein domains, it is possible to replace the enzyme’s native helix-turn-helix DNA-binding motif with a zinc finger DNA-binding motif (Akopian 1999).Cell migration and activation in this process is mediated by signaling molecules known as chemokines (chemoattractant cytokines) which are secreted early in the immune response (de Silva and Stumpf 2004).There are four subfamilies of chemokines and their respective receptors, distinguished by patterns of cysteine residues (abbreviated “C”) in the former.