Hemophilia A, a severe genetic bleeding disorder affecting 1 in 10,000 people, is caused by genetic mutations inducing an inability to produce a clotting factor necessary to stop bleeding. Current treatment—replacing the missing clotting factor (FVIII)—carries risks like costly long-term injections and inhibitor antibodies in ~30% of patients. Our lab investigates base editing, a promising gene therapy solution aiming to repair the mutated FVIII gene directly to recover the natural production of the clotting factor. Our project uses a dog with a naturally occurring mutation similar to human hemophilia A, as a model for developing the treatment. In Phase 1 of this project, I will use mutagenesis and gel electrophoresis to create a plasmid containing the mutant canine FVIII gene, and other lab members will use hydrodynamic injection to apply it into mouse models. Following the verification of delivery through aPTT phenotypic examination, I will begin phase 2 by developing the CRISPR-Cas9 base editing plasmid. This will include insertion of our sgRNA into an existing plasmid, insertion of CAG promoter, maxiprep to produce a large quantity, and finally gel purification of the DNA. Following confirmed DNA sequencing of the constructs, we will use hydrodynamic injection of a mixture of the mutant canine FVIII plasmid and base editing plasmid into our hemophilia A mouse models and aPTT examination to determine the efficacy of the designed plasmid. Phase 3 will apply the base editing plasmid to treat the dog model, through collaboration with other lab members developing delivery strategies. This research holds immense potential for hemophilia A patients, offering a one-time, potentially curative solution compared to the limitations of current treatments. If successful, we expect the project to demonstrate a recovered FVIII gene and FVIII expression- first in the mouse model, then in the canine.

The HIV-1 gag polyprotein consists of the core structural proteins of the virus. During the late stages of viral replication, gag assembles beneath the plasma membrane into a curved immature hexagonal lattice. This process is primarily mediated by the capsid (CA) and spacer peptide 1 (SP1) domains which form a six-helix bundle with the assistance of the naturally occurring small molecule inositol hexakisphosphate (IP₆). Following budding of the immature virion, the HIV-1 protease cleaves the gag domains and CA protein assembles into a mature conical capsid, the protective shell of the virus. Disruption of CA assembly has been shown to inhibit viral replication and in turn has led to the development of the novel antiretroviral drug lenacapavir. While lenacapavir can bind mature capsid cores, the exact mode in which this drug impacts viral assembly and specifically interacts with immature CA remains unclear. My project aims to investigate the effect of lenacapavir and chemically and structurally related analogues of this drug, which have been designed and synthesized by a collaborator, on immature CA assembly in vitro. For this study I optimized the expression and purification of a gag construct that spans the CA to NC domains with an additional N-terminal Serine residue (s-CANC). I transformed E. coli cells with a plasmid containing the s-CANC construct and then overexpressed the protein. I optimized the purification protocol involving gel filtration, ion exchange and immobilized metal affinity chromatography. The purified s-CANC was then used to perform assembly assays using IP₆ in the absence and presence of lenacapavir and the analogues. The formation of immature capsid cores was confirmed via negative staining electron microscopy. Insights from these studies aim to provide a better understanding of how lenacapavir impacts the assembly of immature CA in addition to aiding the development of new capsid targeting antiretroviral drugs.

The HIV-1 capsid is a conical lattice comprised of over 1500 monomeric capsid proteins (CA) that acts as a protective casing for the viral genome. Assembly of the capsid core is driven by the small negatively charged molecule, inositol phosphate (IP6), which interacts with positively charged CA residues. The ability to inhibit viral replication through the disruption of CA assembly has been demonstrated by the novel capsid binding antiretroviral, lenacapavir. Antiretroviral research is also being done on “clickable” compounds which should covalently interact with CA through a Sulfur (VI) Fluoride Exchange (SuFEx) reaction. Our lab has received lenacapavir analogues as well as compounds that potentially interact with CA via a SuFEx reaction (SuFEx compounds). My aim is to assess the impact of lenacapavir and the synthesized analogues on CA assembly, in addition to validating the ability of 15 promising SuFEx compounds to covalently bind CA. In preparation, I transformed E. coli cells with a plasmid containing the HIV-1 CA sequence and induced expression using IPTG. The cells were then pelleted, lysed and the protein purified utilizing gel filtration, anion- and cation-exchange chromatography. Assembly of the purified CA was induced in vitro by the addition of IP6 in both the absence and presence of lenacapavir and the analogues. Relative to lenacapavir, two analogues promoted assembly to a greater extent, while one performed on par and three enhanced assembly to a lesser extent. These analogues will be further studied to determine antiviral activity. Samples of CA in the presence of the SuFEx compounds have also been prepared and sent for mass spectrometry analysis. It will then be determined which of the SuFEx compounds bind CA covalently in vitro and may aid in the development of new capsid targeting antiretrovirals.