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.

Optimizing Integration and Expression of Transgenic Bruton's Tyrosine Kinase for CRISPR-Cas9-Mediated Gene Editing of X-Linked Agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is a monogenic primary immune deficiency characterized by very low levels of immunoglobulins and greatly increased risks for recurrent and severe infections. Patients with XLA have a loss-of-function mutation in the Bruton's tyrosine kinase (BTK) gene and fail to produce mature B lymphocytes. Gene editing in the hematopoietic stem cells of XLA patients to correct or replace the defective gene should restore B cell development and the humoral immune response. We used the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 platform to precisely target integration of a corrective, codon-optimized BTK complementary DNA (cDNA) cassette into its endogenous locus. This process is driven by homologous recombination and should place the transgenic BTK under transcriptional control of its endogenous regulatory elements. Each integrated copy of this cDNA in BTK-deficient K562 cells produced only 11% as much BTK protein as the wild-type gene. The donor cDNA was modified to include the terminal intron of the BTK gene. Successful integration of the intron-containing BTK donor led to a nearly twofold increase in BTK expression per cell over the base donor. However, this donor variant was too large to package into an adeno-associated viral vector for delivery into primary cells. Donors containing truncated variants of the terminal intron also produced elevated expression, although to a lesser degree than the full intron. Addition of the Woodchuck hepatitis virus posttranscriptional regulatory element led to a large boost in BTK transgene expression. Combining these modifications led to a BTK donor template that generated nearly physiological levels of BTK expression in cell lines. These reagents were then optimized to maximize integration rates into human hematopoietic stem and progenitor cells, which have reached potentially therapeutic levels in vitro. The novel donor modifications support effective gene therapy for XLA and will likely assist in the development of other gene editing-based therapies for genetic disorders.

Limiting Cholesterol Biosynthetic Flux Spontaneously Engages Type I IFN Signaling.

Cellular lipid requirements are achieved through a combination of biosynthesis and import programs. Using isotope tracer analysis, we show that type I interferon (IFN) signaling shifts the balance of these programs by decreasing synthesis and increasing import of cholesterol and long chain fatty acids. Genetically enforcing this metabolic shift in macrophages is sufficient to render mice resistant to viral challenge, demonstrating the importance of reprogramming the balance of these two metabolic pathways in vivo. Unexpectedly, mechanistic studies reveal that limiting flux through the cholesterol biosynthetic pathway spontaneously engages a type I IFN response in a STING-dependent manner. The upregulation of type I IFNs was traced to a decrease in the pool size of synthesized cholesterol and could be inhibited by replenishing cells with free cholesterol. Taken together, these studies delineate a metabolic-inflammatory circuit that links perturbations in cholesterol biosynthesis with activation of innate immunity.