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's Tyrosine Kinase (BTK) that mediates the maturation of B cells in the bone marrow and their activation in the periphery. Here we report 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, we employed a CRISPR/Cas9 system that creates a DNA double strand break in an early exon of the BTK locus and an AAV6 virus that carries the donor template for homology directed repair. We evaluated the efficacy of the gene editing approach in HSPCs from XLA patients 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 XLA patients. Using this system, we 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, our work demonstrates the potential of this strategy as a future definitive therapy for XLA

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

Outcomes following SARS-CoV-2 infection in patients with primary and secondary immunodeficiency in the UK

In March 2020, the United Kingdom Primary Immunodeficiency Network (UKPIN) established a registry of cases to collate the outcomes of individuals with PID and SID following SARS-CoV-2 infection and treatment. A total of 310 cases of SARS-CoV-2 infection in individuals with PID or SID have now been reported in the UK. The overall mortality within the cohort was 17.7% (n = 55/310). Individuals with CVID demonstrated an infection fatality rate (IFR) of 18.3% (n = 17/93), individuals with PID receiving IgRT had an IFR of 16.3% (n = 26/159) and individuals with SID, an IFR of 27.2% (n = 25/92). Individuals with PID and SID had higher inpatient mortality and died at a younger age than the general population. Increasing age, low pre-SARS-CoV-2 infection lymphocyte count and the presence of common co-morbidities increased the risk of mortality in PID. Access to specific COVID-19 treatments in this cohort was limited: only 22.9% (n = 33/144) of patients admitted to the hospital received dexamethasone, remdesivir, an anti-SARS-CoV-2 antibody-based therapeutic (e.g. REGN-COV2 or convalescent plasma) or tocilizumab as a monotherapy or in combination. Dexamethasone, remdesivir, and anti-SARS-CoV-2 antibody-based therapeutics appeared efficacious in PID and SID. Compared to the general population, individuals with PID or SID are at high risk of mortality following SARS-CoV-2 infection. Increasing age, low baseline lymphocyte count, and the presence of co-morbidities are additional risk factors for poor outcome in this cohort

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

Evidence that a STAT3 Mutation Causing Hyper IgE Syndrome Leads to Repression of Transcriptional Activity

We present the case of a 19-year-old female with a mild form of Autosomal Dominant Hyper IgE syndrome (HIES) associated with a loss-of-function mutation in STAT3. Within the first years of life she developed multiple, Staphylococcus aureus associated abscesses in the neck and face requiring frequent incision and drainage. Respiratory tract infections were not a feature of the clinical phenotype and a high resolution thoracic CT scan was unremarkable. Retained dentition was noted but fungal nail disease and recurrent thrush were absent. The total IgE was 970 IU/L, Lymphocyte counts and immunoglobulin levels were normal (IgG borderline 18.5 gr/L). There was suboptimal response to test immunisation with Pneumovax II vaccine. Th17 cell phenotyping revealed low levels of IL-17 expressing cells (0.3% of total CD4 T Cells numbers). Genetic analysis identified a missense mutation, N567D, in a conserved region of the linker domain of STAT3. Functional studies in HEK293 cells reveal that this mutation potently inhibits STAT3 activity when compared to the wildtype protein. This is consistent with other reported mutations in STAT3 associated with HIES. However, surprisingly, the magnitude of inhibition was similar to another STAT3 mutation (V637M) which causes a much more severe form of the disease

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 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 optimisation process is laborious and not always clearly defined. We have developed a flexible multidimensional Response Surface Methodology approach to optimisation of gene correction to rapidly investigate and select editing conditions for CD34+ cells with the best possible balance between correction and cell/CFU loss in a parsimonious one-shot experiment. This method revealed that using 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 optimised editing conditions for the correction of p67phox-deficient chronic granulomatous disease, an autosomal recessive disorder of blood phagocytic cells resulting in severe recurrent bacterial and fungal infections, allowing rescue of p67phox expression and functional correction of CD34+-derived neutrophils from a CGD patient

COVID-19 in patients with primary and secondary immunodeficiency: The United Kingdom experience

Background As of November 2020, severe acute respiratory syndrome coronavirus 2 has resulted in 55 million infections worldwide and more than 1.3 million deaths from coronavirus disease 2019 (COVID-19). Outcomes following severe acute respiratory syndrome coronavirus 2 infection in individuals with primary immunodeficiency (PID) or symptomatic secondary immunodeficiency (SID) remain uncertain. Objectives We sought to document the outcomes of individuals with PID or symptomatic SID following COVID-19 in the United Kingdom. Methods At the start of the COVID-19 pandemic, the United Kingdom Primary Immunodeficiency Network established a registry of cases to collate the nationwide outcomes of COVID-19 in individuals with PID or symptomatic SID and determine risk factors associated with morbidity and mortality from COVID-19 in these patient groups. Results A total of 100 patients had been enrolled by July 1, 2020, 60 with PID, 7 with other inborn errors of immunity including autoinflammatory diseases and C1 inhibitor deficiency, and 33 with symptomatic SID. In individuals with PID, 53.3% (32 of 60) were hospitalized, the infection-fatality ratio was 20.0% (12 of 60), the case-fatality ratio was 31.6% (12 of 38), and the inpatient mortality was 37.5% (12 of 32). Individuals with SID had worse outcomes than those with PID; 75.8% (25 of 33) were hospitalized, the infection-fatality ratio was 33.3% (11 of 33), the case-fatality ratio was 39.2% (11 of 28), and inpatient mortality was 44.0% (11 of 25). Conclusions In comparison to the general population, adult patients with PID and symptomatic SID display greater morbidity and mortality from COVID-19. This increased risk must be reflected in public health guidelines to adequately protect vulnerable patients from exposure to the virus

Outcomes following SARS-CoV-2 infection in patients with primary and secondary immunodeficiency in the UK.

In March 2020, the United Kingdom Primary Immunodeficiency Network (UKPIN) established a registry of cases to collate the outcomes of individuals with PID and SID following SARS-CoV-2 infection and treatment. A total of 310 cases of SARS-CoV-2 infection in individuals with PID or SID have now been reported in the UK. The overall mortality within the cohort was 17.7% (n = 55/310). Individuals with CVID demonstrated an infection fatality rate (IFR) of 18.3% (n = 17/93), individuals with PID receiving IgRT had an IFR of 16.3% (n = 26/159) and individuals with SID, an IFR of 27.2% (n = 25/92). Individuals with PID and SID had higher inpatient mortality and died at a younger age than the general population. Increasing age, low pre-SARS-CoV-2 infection lymphocyte count and the presence of common co-morbidities increased the risk of mortality in PID. Access to specific COVID-19 treatments in this cohort was limited: only 22.9% (n = 33/144) of patients admitted to the hospital received dexamethasone, remdesivir, an anti-SARS-CoV-2 antibody-based therapeutic (e.g. REGN-COV2 or convalescent plasma) or tocilizumab as a monotherapy or in combination. Dexamethasone, remdesivir, and anti-SARS-CoV-2 antibody-based therapeutics appeared efficacious in PID and SID. Compared to the general population, individuals with PID or SID are at high risk of mortality following SARS-CoV-2 infection. Increasing age, low baseline lymphocyte count, and the presence of co-morbidities are additional risk factors for poor outcome in this cohort