Genes as Medicine: The Development of Gene Therapies for Inborn Errors of Immunity.

The field of gene therapy has experienced tremendous growth in the last decade ranging from improvements in the design of viral vectors for gene addition of therapeutic gene cassettes to the discovery of site-specific nucleases targeting transgenes to desired locations in the genome. Such advancements have not only enabled the development of disease models but also created opportunities for the development of tailored therapeutic approaches. There are 3 main methods of gene modification that can be used for the prevention or treatment of disease. This includes viral vector-mediated gene therapy to supply or bypass a missing/defective gene, gene editing enabled by programmable nucleases to create sequence-specific alterations in the genome, and gene silencing to reduce the expression of a gene or genes. These gene-modification platforms can be delivered either in vivo, for which the therapy is injected directed into a patient's body, or ex vivo, in which cells are harvested from a patient and modified in a laboratory setting, and then returned to the patient.

A comparison of DNA repair pathways to achieve a site-specific gene modification of the Bruton's tyrosine kinase gene.

Gene editing utilizing homology-directed repair has advanced significantly for many monogenic diseases of the hematopoietic system in recent years but has also been hindered by decreases between in vitro and in vivo gene integration rates. Homology-directed repair occurs primarily in the S/G2 phases of the cell cycle, whereas long-term engrafting hematopoietic stem cells are typically quiescent. Alternative methods for a targeted integration have been proposed including homology-independent targeted integration and precise integration into target chromosome, which utilize non-homologous end joining and microhomology-mediated end joining, respectively. Non-homologous end joining occurs throughout the cell cycle, while microhomology-mediated end joining occurs predominantly in the S phase. We compared these pathways for the integration of a corrective DNA cassette at the Bruton's tyrosine kinase gene for the treatment of X-linked agammaglobulinemia. Homology-directed repair generated the most integration in K562 cells; however, synchronizing cells into G1 resulted in the highest integration rates with homology-independent targeted integration. Only homology-directed repair produced seamless junctions, making it optimal for targets where insertions and deletions are impermissible. Bulk CD34+ cells were best edited by homology-directed repair and precise integration into the target chromosome, while sorted hematopoietic stem cells contained similar integration rates using all corrective donors.

The Stapled AKAP Disruptor Peptide STAD-2 Displays Antimalarial Activity through a PKA-Independent Mechanism.

Drug resistance poses a significant threat to ongoing malaria control efforts. Coupled with lack of a malaria vaccine, there is an urgent need for the development of new antimalarials with novel mechanisms of action and low susceptibility to parasite drug resistance. Protein Kinase A (PKA) has been implicated as a critical regulator of pathogenesis in malaria. Therefore, we sought to investigate the effects of disrupted PKA signaling as a possible strategy for inhibition of parasite replication. Host PKA activity is partly regulated by a class of proteins called A Kinase Anchoring Proteins (AKAPs), and interaction between HsPKA and AKAP can be inhibited by the stapled peptide Stapled AKAP Disruptor 2 (STAD-2). STAD-2 was tested for permeability to and activity against Plasmodium falciparum blood stage parasites in vitro. The compound was selectively permeable only to infected red blood cells (iRBC) and demonstrated rapid antiplasmodial activity, possibly via iRBC lysis (IC50 ≈ 1 µM). STAD-2 localized within the parasite almost immediately post-treatment but showed no evidence of direct association with PKA, indicating that STAD-2 acts via a PKA-independent mechanism. Furosemide-insensitive parasite permeability pathways in the iRBC were largely responsible for uptake of STAD-2. Further, peptide import was highly specific to STAD-2 as evidenced by low permeability of control stapled peptides. Selective uptake and antiplasmodial activity of STAD-2 provides important groundwork for the development of stapled peptides as potential antimalarials. Such peptides may also offer an alternative strategy for studying protein-protein interactions critical to parasite development and pathogenesis.