What a Clinician Needs to Know About Genome Editing: Status and Opportunities for Inborn Errors of Immunity.

During the past 20 years, gene editing has emerged as a novel form of gene therapy. Since the publication of the first potentially therapeutic gene editing platform for genetic disorders, increasingly sophisticated editing technologies have been developed. As with viral vector-mediated gene addition, inborn errors of immunity are excellent candidate diseases for a corrective autologous hematopoietic stem cell gene editing strategy. Research on gene editing for inborn errors of immunity is still entirely preclinical, with no trials yet underway. However, with editing techniques maturing, scientists are investigating this novel form of gene therapy in context of an increasing number of inborn errors of immunity. Here, we present an overview of these studies and the recent progress moving these technologies closer to clinical benefit.

Immunotherapy responsive neuroinflammation in a child with FAS-associated death-domain mutation.

Death-inducing signaling complex (DISC), FAS-associated death-domain-containing protein (FADD), Fas receptor (FASR), Fas ligand (FASL).

Lentiviral Gene Therapy for Artemis-Deficient SCID.

BACKGROUND: The DNA-repair enzyme Artemis is essential for rearrangement of T- and B-cell receptors. Mutations in DCLRE1C, which encodes Artemis, cause Artemis-deficient severe combined immunodeficiency (ART-SCID), which is poorly responsive to allogeneic hematopoietic-cell transplantation. METHODS: We carried out a phase 1-2 clinical study of the transfusion of autologous CD34+ cells, transfected with a lentiviral vector containing DCLRE1C, in 10 infants with newly diagnosed ART-SCID. We followed them for a median of 31.2 months. RESULTS: Marrow harvest, busulfan conditioning, and lentiviral-transduced CD34+ cell infusion produced the expected grade 3 or 4 adverse events. All the procedures met prespecified criteria for feasibility at 42 days after infusion. Gene-marked T cells were detected at 6 to 16 weeks after infusion in all the patients. Five of 6 patients who were followed for at least 24 months had T-cell immune reconstitution at a median of 12 months. The diversity of T-cell receptor ß chains normalized by 6 to 12 months. Four patients who were followed for at least 24 months had sufficient B-cell numbers, IgM concentration, or IgM isohemagglutinin titers to permit discontinuation of IgG infusions. Three of these 4 patients had normal immunization responses, and the fourth has started immunizations. Vector insertion sites showed no evidence of clonal expansion. One patient who presented with cytomegalovirus infection received a second infusion of gene-corrected cells to achieve T-cell immunity sufficient for viral clearance. Autoimmune hemolytic anemia developed in 4 patients 4 to 11 months after infusion; this condition resolved after reconstitution of T-cell immunity. All 10 patients were healthy at the time of this report. CONCLUSIONS: Infusion of lentiviral gene-corrected autologous CD34+ cells, preceded by pharmacologically targeted low-exposure busulfan, in infants with newly diagnosed ART-SCID resulted in genetically corrected and functional T and B cells. (Funded by the California Institute for Regenerative Medicine and the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT03538899.).

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.

Outcomes following treatment for ADA-deficient severe combined immunodeficiency: a report from the PIDTC.

Adenosine deaminase (ADA) deficiency causes ∼13% of cases of severe combined immune deficiency (SCID). Treatments include enzyme replacement therapy (ERT), hematopoietic cell transplant (HCT), and gene therapy (GT). We evaluated 131 patients with ADA-SCID diagnosed between 1982 and 2017 who were enrolled in the Primary Immune Deficiency Treatment Consortium SCID studies. Baseline clinical, immunologic, genetic characteristics, and treatment outcomes were analyzed. First definitive cellular therapy (FDCT) included 56 receiving HCT without preceding ERT (HCT); 31 HCT preceded by ERT (ERT-HCT); and 33 GT preceded by ERT (ERT-GT). Five-year event-free survival (EFS, alive, no need for further ERT or cellular therapy) was 49.5% (HCT), 73% (ERT-HCT), and 75.3% (ERT-GT; P < .01). Overall survival (OS) at 5 years after FDCT was 72.5% (HCT), 79.6% (ERT-HCT), and 100% (ERT-GT; P = .01). Five-year OS was superior for patients undergoing HCT at <3.5 months of age (91.6% vs 68% if ≥3.5 months, P = .02). Active infection at the time of HCT (regardless of ERT) decreased 5-year EFS (33.1% vs 68.2%, P < .01) and OS (64.7% vs 82.3%, P = .02). Five-year EFS (90.5%) and OS (100%) were best for matched sibling and matched family donors (MSD/MFD). For patients treated after the year 2000 and without active infection at the time of FDCT, no difference in 5-year EFS or OS was found between HCT using a variety of transplant approaches and ERT-GT. This suggests alternative donor HCT may be considered when MSD/MFD HCT and GT are not available, particularly when newborn screening identifies patients with ADA-SCID soon after birth and before the onset of infections. This trial was registered at www.clinicaltrials.gov as #NCT01186913 and #NCT01346150.

Ocular findings associated with FADD deficiency resemble familial exudative vitreoretinopathy.

PURPOSE: We report the first known case of eye findings associated with a Fas-associated protein with death domain (FADD) gene mutation, an exceedingly rare entity. OBSERVATIONS: A 7-year-old boy was referred for decreased vision and eye examination revealed cystoid macular edema and peripheral retinal ischemia in both eyes and progression to tractional retinal detachment in the right eye. CONCLUSIONS AND IMPORTANCE: This case suggests that baseline and annual ophthalmic screening may be beneficial in individuals with FADD mutations. However, greater documentation of cases may be necessary before deriving a clear interval screening recommendation.

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.

Adenosine Deaminase (ADA)-Deficient Severe Combined Immune Deficiency (SCID) in the US Immunodeficiency Network (USIDNet) Registry.

Clinical data from ADA-SCID patients registered in the U.S. Immunodeficiency Network (USIDNet) Repository were analyzed. Sixty-four ADA-SCID patients born between 1981 and 2017 had clinical data entered by their local (or home) enrolling institution. Median age at diagnosis was 1 month for those with a positive family history and 3 months for those without a prior family history, with some diagnosed at birth and one as late as 9 years of age. Overall survival was 79.7%, which increased to 94.1% since 2010. These patients had multiple infections and pulmonary, gastrointestinal, and neurological complications. The majority received enzyme replacement therapy (ERT) at some time, including 88% of those born since 2010. Twenty-six patients underwent allogeneic hematopoietic stem cell transplant (HSCT). HSCT successfully supported survival (17/26, 65%) using a variety of cell sources (bone marrow, mobilized peripheral blood, and cord blood) from sibling, family and unrelated donors. Nineteen patients underwent autologous HSCT with gene therapy (GT) using retroviral and lentiviral vectors and all are surviving. The prognosis for patients with ADA-SCID has continued to improve but these patients do have multiple early and potentially long-term conditions that require medical monitoring and management.

Overview of the current status of gene therapy for primary immune deficiencies (PIDs).

Over 3 decades, gene therapy has advanced from a logical idea to becoming a clinical reality for several of the most severe primary immune deficiencies, as well as other inherited disorders. The first gene therapy medicines have been licensed for marketing and several more are advancing toward that goal to make them widely available, beyond clinical trials. Although common platforms of cells, vectors, or editing reagents are used for these disorders, each individual genetic cause of an immune deficiency requires its own vector or editing tools and a package of preclinical data on efficacy and safety to initiate clinical trials. One-by-one, gene therapy for primary immune deficiencies is being brought to the clinic and hopefully will provide safe and effective therapies.

Lentiviral gene therapy for X-linked chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a rare inherited disorder of phagocytic cells1,2. We report the initial results of nine severely affected X-linked CGD (X-CGD) patients who received ex vivo autologous CD34+ hematopoietic stem and progenitor cell-based lentiviral gene therapy following myeloablative conditioning in first-in-human studies (trial registry nos. NCT02234934 and NCT01855685). The primary objectives were to assess the safety and evaluate the efficacy and stability of biochemical and functional reconstitution in the progeny of engrafted cells at 12 months. The secondary objectives included the evaluation of augmented immunity against bacterial and fungal infection, as well as assessment of hematopoietic stem cell transduction and engraftment. Two enrolled patients died within 3 months of treatment from pre-existing comorbidities. At 12 months, six of the seven surviving patients demonstrated stable vector copy numbers (0.4-1.8 copies per neutrophil) and the persistence of 16-46% oxidase-positive neutrophils. There was no molecular evidence of either clonal dysregulation or transgene silencing. Surviving patients have had no new CGD-related infections, and six have been able to discontinue CGD-related antibiotic prophylaxis. The primary objective was met in six of the nine patients at 12 months follow-up, suggesting that autologous gene therapy is a promising approach for CGD patients.

Editing the Sickle Cell Disease Mutation in Human Hematopoietic Stem Cells: Comparison of Endonucleases and Homologous Donor Templates.

Site-specific correction of a point mutation causing a monogenic disease in autologous hematopoietic stem and progenitor cells (HSPCs) can be used as a treatment of inherited disorders of the blood cells. Sickle cell disease (SCD) is an ideal model to investigate the potential use of gene editing to transvert a single point mutation at the ß-globin locus (HBB). We compared the activity of zinc-finger nucleases (ZFNs) and CRISPR/Cas9 for editing, and homologous donor templates delivered as single-stranded oligodeoxynucleotides (ssODNs), adeno-associated virus serotype 6 (AAV6), integrase-deficient lentiviral vectors (IDLVs), and adenovirus 5/35 serotype (Ad5/35) to transvert the base pair responsible for SCD in HBB in primary human CD34+ HSPCs. We found that the ZFNs and Cas9 directed similar frequencies of nuclease activity. In vitro, AAV6 led to the highest frequencies of homology-directed repair (HDR), but levels of base pair transversions were significantly reduced when analyzing cells in vivo in immunodeficient mouse xenografts, with similar frequencies achieved with either AAV6 or ssODNs. AAV6 also caused significant impairment of colony-forming progenitors and human cell engraftment. Gene correction in engrafting hematopoietic stem cells may be limited by the capacity of the cells to mediate HDR, suggesting additional manipulations may be needed for high-efficiency gene correction in HSPCs.

Immune and Genetic Features of the Chromosome 22q11.2 Deletion (DiGeorge Syndrome).

PURPOSE OF REVIEW: This review provides an update on the progress in identifying the range of immunological dysfunction seen in DiGeorge syndrome and on more recent diagnostic and treatment approaches. RECENT FINDINGS: Clinically, the associated thymic hypoplasia/aplasia is well known and can have profound effects on T cell function. Further, the humoral arm of the immune system can be affected, with hypogammaglobulinemia and poor vaccine-specific antibody response. Additionally, genetic testing utilizing chromosomal microarray demonstrates a small but significant number of 22q11 deletions that are not detectable by standard FISH testing. The recent addition of a TREC assay to newborn screening can identify a subset of infants whose severe immune defects may result from 22q11 deletion. This initial presentation now also places the immunologist in the role of "first responder" with regard to diagnosis and management of these patients. DiGeorge syndrome reflects a clinical phenotype now recognized by its underlying genetic diagnosis, chromosome 22q11.2 deletion syndrome, which is associated with multisystem involvement and variable immune defects among patients. Updated genetic and molecular techniques now allow for earlier identification of immune defects and confirmatory diagnoses, in this disorder with life-long clinical issues.

Advances in site-specific gene editing for primary immune deficiencies.

PURPOSE OF REVIEW: Conventional gene therapy has been a successful, curative treatment modality for many primary immune deficiencies with significant improvements in the last decade. However, the risk of leukemic transformation with viral-mediated gene addition still remains, and unregulated gene addition is not an option for certain diseases in which the target gene is closely controlled. The recent bloom in genome modification platforms has created the opportunity to site-specifically correct mutated DNA base pairs or insert a corrective cDNA minigene while maintaining gene expression under control of endogenous regulatory elements. RECENT FINDINGS: There is an abundance of ongoing research utilizing programmable nucleases to facilitate site-specific gene correction of many primary immune deficiencies including X-linked severe combined immune deficiency, X-linked chronic granulomatous disease, Wiskott-Aldrich syndrome, X-linked hyper-IgM syndrome, X-linked agammaglobulinemia, and immune dysregulation, polyendocrinopathy, enteropathy, X-linked. In all, these studies have demonstrated the ability to integrate corrective DNA sequences at a precise location in the genome at rates likely to either cure or ameliorate disease. SUMMARY: Gene editing for primary immune deficiency (PID) has advanced to the point to that translation to clinical trials is likely to occur in the next several years. At the current pace of research in DNA repair mechanisms, stem cell biology, and genome-editing technology, targeted genome modification represents the next chapter of gene therapy for PID.

The PTH/PTHrP-SIK3 pathway affects skeletogenesis through altered mTOR signaling.

Studies have suggested a role for the mammalian (or mechanistic) target of rapamycin (mTOR) in skeletal development and homeostasis, yet there is no evidence connecting mTOR with the key signaling pathways that regulate skeletogenesis. We identified a parathyroid hormone (PTH)/PTH-related peptide (PTHrP)-salt-inducible kinase 3 (SIK3)-mTOR signaling cascade essential for skeletogenesis. While investigating a new skeletal dysplasia caused by a homozygous mutation in the catalytic domain of SIK3, we observed decreased activity of mTOR complex 1 (mTORC1) and mTORC2 due to accumulation of DEPTOR, a negative regulator of both mTOR complexes. This SIK3 syndrome shared skeletal features with Jansen metaphyseal chondrodysplasia (JMC), a disorder caused by constitutive activation of the PTH/PTHrP receptor. JMC-derived chondrocytes showed reduced SIK3 activity, elevated DEPTOR, and decreased mTORC1 and mTORC2 activity, indicating a common mechanism of disease. The data demonstrate that SIK3 is an essential positive regulator of mTOR signaling that functions by triggering DEPTOR degradation in response to PTH/PTHrP signaling during skeletogenesis.

Site-Specific Gene Editing of Human Hematopoietic Stem Cells for X-Linked Hyper-IgM Syndrome.

X-linked hyper-immunoglobulin M (hyper-IgM) syndrome (XHIM) is a primary immunodeficiency due to mutations in CD40 ligand that affect immunoglobulin class-switch recombination and somatic hypermutation. The disease is amenable to gene therapy using retroviral vectors, but dysregulated gene expression results in abnormal lymphoproliferation in mouse models, highlighting the need for alternative strategies. Here, we demonstrate the ability of both the transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) platforms to efficiently drive integration of a normal copy of the CD40L cDNA delivered by Adeno-Associated Virus. Site-specific insertion of the donor sequence downstream of the endogenous CD40L promoter maintained physiologic expression of CD40L while overriding all reported downstream mutations. High levels of gene modification were achieved in primary human hematopoietic stem cells (HSCs), as well as in cell lines and XHIM-patient-derived T cells. Notably, gene-corrected HSCs engrafted in immunodeficient mice at clinically relevant frequencies. These studies provide the foundation for a permanent curative therapy in XHIM.

Characterization of Gene Alterations following Editing of the ß-Globin Gene Locus in Hematopoietic Stem/Progenitor Cells.

The use of engineered nucleases combined with a homologous DNA donor template can result in targeted gene correction of the sickle cell disease mutation in hematopoietic stem and progenitor cells. However, because of the high homology between the adjacent human ß- and δ-globin genes, off-target cleavage is observed at δ-globin when using some endonucleases targeted to the sickle mutation in ß-globin. Introduction of multiple double-stranded breaks by endonucleases has the potential to induce intergenic alterations. Using a novel droplet digital PCR assay and high-throughput sequencing, we characterized the frequency of rearrangements between the ß- and δ-globin paralogs when delivering these nucleases. Pooled CD34+ cells and colony-forming units from sickle bone marrow were treated with nuclease only or including a donor template and then analyzed for potential gene rearrangements. It was observed that, in pooled CD34+ cells and colony-forming units, the intergenic ß-δ-globin deletion was the most frequent rearrangement, followed by inversion of the intergenic fragment, with the inter-chromosomal translocation as the least frequent. No rearrangements were observed when endonuclease activity was restricted to on-target ß-globin cleavage. These findings demonstrate the need to develop site-specific endonucleases with high specificity to avoid unwanted gene alterations.