Hematopoietic stem cell gene editing rescues B-cell development in X-linked agammaglobulinemia.

BACKGROUND: X-linked agammaglobulinemia (XLA) is an inborn error of immunity that renders boys susceptible to life-threatening infections due to loss of mature B cells and circulating immunoglobulins. It is caused by defects in the gene encoding the Bruton tyrosine kinase (BTK) that mediates the maturation of B cells in the bone marrow and their activation in the periphery. This paper reports on a gene editing protocol to achieve "knock-in" of a therapeutic BTK cassette in hematopoietic stem and progenitor cells (HSPCs) as a treatment for XLA. METHODS: To rescue BTK expression, this study employed a clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system that creates a DNA double-strand break in an early exon of the BTK locus and an adeno-associated virus 6 virus that carries the donor template for homology-directed repair. The investigators evaluated the efficacy of the gene editing approach in HSPCs from patients with XLA that were cultured in vitro under B-cell differentiation conditions or that were transplanted in immunodeficient mice to study B-cell output in vivo. RESULTS: A (feeder-free) B-cell differentiation protocol was successfully applied to blood-mobilized HSPCs to reproduce in vitro the defects in B-cell maturation observed in patients with XLA. Using this system, the investigators could show the rescue of B-cell maturation by gene editing. Transplantation of edited XLA HSPCs into immunodeficient mice led to restoration of the human B-cell lineage compartment in the bone marrow and immunoglobulin production in the periphery. CONCLUSIONS: Gene editing efficiencies above 30% could be consistently achieved in human HSPCs. Given the potential selective advantage of corrected cells, as suggested by skewed X-linked inactivation in carrier females and by competitive repopulating experiments in mouse models, this work demonstrates the potential of this strategy as a future definitive therapy for XLA.

Multidimensional Response Surface Methodology for the Development of a Gene Editing Protocol for p67phox-Deficient Chronic Granulomatous Disease.

Replacing a faulty gene with a correct copy has become a viable therapeutic option as a result of recent progress in gene editing protocols. Targeted integration of therapeutic genes in hematopoietic stem cells has been achieved for multiple genes using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system and Adeno-Associated Virus (AAV) to carry a donor template. Although this is a promising strategy to correct genetic blood disorders, it is associated with toxicity and loss of function in CD34+ hematopoietic stem and progenitor cells, which has hampered clinical application. Balancing the maximum achievable correction against deleterious effects on the cells is critical. However, multiple factors are known to contribute, and the optimization process is laborious and not always clearly defined. We have developed a flexible multidimensional Response Surface Methodology approach for optimization of gene correction. Using this approach, we could rapidly investigate and select editing conditions for CD34+ cells with the best possible balance between correction and cell/colony-forming unit (CFU) loss in a parsimonious one-shot experiment. This method revealed that using relatively low doses of AAV2/6 and CRISPR/Cas9 ribonucleoprotein complex, we can preserve the fitness of CD34+ cells and, at the same time, achieve high levels of targeted gene insertion. We then used these optimized editing conditions for the correction of p67phox-deficient chronic granulomatous disease (CGD), an autosomal recessive disorder of blood phagocytic cells resulting in severe recurrent bacterial and fungal infections and achieved rescue of p67phox expression and functional correction of CD34+-derived neutrophils from a CGD patient.

Gene Editing in Human Haematopoietic Stem Cells for the Treatment of Primary Immunodeficiencies.

In recent years, gene-editing technologies have revolutionised precision medicine, and human trials of this technology have been reported in cell-based cancer therapies and other genetic disorders. The same techniques have the potential to reverse mutations in monogenic primary immunodeficiencies (PIDs), and transplantation of edited haematopoietic stem cells may provide a functional cure for these diseases. In this review, we discuss the methods of gene editing being explored and describe progress made so far with several PIDs. We also detail the remaining challenges, how to confidently detect off-target effects and chromosomal abnormalities in a timely manner, how to obtain long-term benefits, and how to achieve physiological levels of expression of the therapeutic gene. With advances in gene editing, we envisage a robust clinical translation of this technology in the coming decade.

Comparison of gastrojejunal anastomosis techniques in laparoscopic Roux-en-Y gastric bypass: gastrojejunal stricture rate and effect on subsequent weight loss.

BACKGROUND: Different gastrojejunal anastomotic (GJA) techniques have been described in laparoscopic Roux-en-Y gastric bypass (LRYGB). There is conflicting data on whether one technique is superior to the other. We aimed to compare hand-sewn (HSA), circular-stapled (CSA) and linear-stapled (LSA) anastomotic techniques in terms of stricture rates and their impact on subsequent weight loss. METHODS: A prospectively collected database was used to identify patients undergoing LRYGB surgery between March 2005 and May 2012. Anastomotic technique (HSA, CSA, LSA) was performed according to individual surgeon preference. The database recorded patient demographics, relevant comorbidities and the type of GJA performed. Serial weight measurements and percentage excess weight loss (%EWL) were available at defined follow-up intervals. RESULTS: Included in the data were 426 patients, divided between HSA (n = 174, 40.8%), CSA (n = 110, 25.8%) and LSA (n = 142, 33.3%). There was no significant difference in the stricture rates (HSA n = 17, 9.72%; CSA n = 9, 8.18%; LSA n = 8, 5.63%; p = 0.4006). Weight loss was similar between the three techniques (HSA, CSA and LSA) at 3 months (40.6% ± 16.2% vs 35.92% ± 21.42% vs 48.21 % ± 14.79%; p = 0.0821), 6 months (61.48% ± 23.94% vs 58.16 % ± 27.31% vs 60.18% ± 22.26%; p = 0.2296), 12 months (72.94% ± 19.93% vs 69.72 ± 21.42% vs 66.05% ± 17.75%; p = 0.0617) and 24 months (73.29% ± 22.31% vs 68.75 % ± 24.71% vs 69.40% ± 23.10%; p = 0.7242), respectively. The stricture group lost significantly greater weight (%EWL) within the first 3 months compared to the non-stricture group (45.39% ± 16.82 % vs 39.22 % ± 21.93%; p = 0.0340); however, this difference had resolved at 6 months (61.29% ± 18.50 % vs 59.79% ± 23.03%; p = 0.8802) and 12 months (71.59 % ± 18.67 % vs 68.69 % ± 22.19 %; p = 0.5970). CONCLUSIONS: There was no significant difference in the rate of strictures between the three techniques, although the linear technique appears to have the lowest requirement for post-operative dilatation. The re-intervention rate will, in part, be dictated by the threshold for endoscopy, which will vary between units. Weight loss was similar between the three anastomotic techniques. Surgeons should use techniques that they are most familiar with, as stricture and weight loss rates are not significantly different.