The most severe primary immune deficiency diseases (PIDs) have been successfully treated with allogeneic hematopoietic stem cell transplantation for more than 4 decades. However, such transplantations have the best outcomes when there is a well-matched donor available because immune complications, such as graft-versus-host disease, are greater without a matched sibling donor. Gene therapy has been developed as a method to perform autologous transplantations of a patient's own stem cells that are genetically corrected. Through an iterative bench-to-bedside-and-back process, methods to efficiently add new copies of the relevant gene to hematopoietic stem cells have led to safe and effective treatments for several PIDs, including forms of severe combined immune deficiency, Wiskott-Aldrich syndrome, and chronic granulomatous disease. New methods for gene editing might allow additional PIDs to be treated by gene therapy because they will allow the endogenous gene to be repaired and expressed under its native regulatory elements, which are essential for genes involved in cell processes of signaling, activation, and proliferation. Gene therapy is providing exciting new treatment options for patients with PIDs, and advances are sure to continue.

The use of gene therapy in the treatment of primary immune deficiencies (PID) has advanced significantly in the last decade. Clinical trials for X-linked severe combined immunodeficiency, adenosine deaminase deficiency (ADA), chronic granulomatous disease, and Wiskott-Aldrich syndrome have demonstrated that gene transfer into hematopoietic stem cells and autologous transplant can result in clinical improvement and is curative for many patients. Unfortunately, early clinical trials were complicated by vector-related insertional mutagenic events for several diseases with the exception of ADA-deficiency SCID. These results prompted the current wave of clinical trials for primary immunodeficiency using alternative retro- or lenti-viral vector constructs that are self-inactivating, and they have shown clinical efficacy without leukemic events thus far. The field of gene therapy continues to progress, with improvements in viral vector profiles, stem cell culturing techniques, and site-specific genome editing platforms. The future of gene therapy is promising, and we are quickly moving towards a time when it will be a standard cellular therapy for many forms of PID.

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.

Recognition of non-SCID and secondary causes of T cell lymphopenia detected by TREC newborn screening is important in directing subsequent care and identifying those who would not benefit from more invasive interventions. Here, we report two infants with low TRECs and severe, but self-resolving, T cell lymphopenia identified by SCID NBS that were caused by in utero exposure to purine antimetabolites.

In the United States, newborn screening (NBS) is currently recommended for identification of 31 debilitating and potentially fatal conditions. However, individual states determine which of the recommended conditions are screened. The addition of severe combined immunodeficiency (SCID) screening to the recommended NBS panel has been fully instituted by 18 states, with another 11 states piloting programs or planning to begin screening in 2014. Untreated, SCID is uniformly fatal by 2 years of age. Hematopoietic stem cell transplantation usually is curative, but the success rate depends on the age at which the procedure is performed. Short-term implementation costs may be a barrier to adding SCID to states' NBS panels. A retrospective economic analysis was performed to determine the cost-effectiveness of NBS for early (<3.5 months) versus late (≥3.5 months) treatment of children with SCID at 3 centers over 5 years. The mean total charges at these centers for late treatment were 4 times greater than early treatment ($1.43 million vs $365,785, respectively). Mean charges for intensive care treatments were >5 times higher ($350,252 vs $66,379), and operating room-anesthesia charges were approximately 4 times higher ($57,105 vs $15,885). The cost-effectiveness of early treatment for SCID provides a strong economic rationale for the addition of SCID screening to NBS programs of other states.

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.

Recombinant adeno-associated viruses (rAAV) are promising for applications in many genome editing techniques through their effectiveness as carriers of DNA homologous donors into primary hematopoietic stem and progenitor cells (HSPCs), but they have many outstanding concerns. Specifically, their biomanufacturing and the variety of factors that influence the quality and consistency of rAAV preps are in question. During the process of rAAV packaging, a cell line is transfected with several DNA plasmids that collectively encode all the necessary information to allow for viral packaging. Ideally, this process results in the packaging of complete viral particles only containing rAAV genomes; however, this is not the case. Through this study, we were able to leverage single-stranded virus (SSV) sequencing, a next-generation sequencing-based method to quantify all DNA species present within rAAV preps. From this, it was determined that much of the DNA within some rAAV preps is not vector-genome derived, and there is wide variability in the contamination by DNA across various preps. Furthermore, we demonstrate that transducing CD34+ HSPCs with preps with higher contaminating DNA resulted in decreased clonogenic potential, altered transcriptomic profiles, and decreased genomic editing. Collectively, this study characterized the effects of DNA contamination within rAAV preps on CD34+ HSPC cellular potential.

We examined the efficiency, specificity, and mutational signatures of zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 systems designed to target the gene encoding the transcriptional repressor BCL11A, in human K562 cells and human CD34⁺ progenitor cells. ZFNs and TALENs were delivered as in vitro transcribed mRNA through electroporation; CRISPR/Cas9 was codelivered by Cas9 mRNA with plasmid-encoded guideRNA (gRNA) (pU6.g1) or in vitro transcribed gRNA (gR.1). Analyses of efficacy revealed that for these specific reagents and the delivery methods used, the ZFNs gave rise to more allelic disruption in the targeted locus compared to the TALENs and CRISPR/Cas9, which was associated with increased levels of fetal hemoglobin in erythroid cells produced in vitro from nuclease-treated CD34⁺ cells. Genome-wide analysis to evaluate the specificity of the nucleases revealed high specificity of this specific ZFN to the target site, while specific TALENs and CRISPRs evaluated showed off-target cleavage activity. ZFN gene-edited CD34⁺ cells had the capacity to engraft in NOD-Prkdc^SCID-IL2Rγ^null mice, while retaining multi-lineage potential, in contrast to TALEN gene-edited CD34⁺ cells. CRISPR engraftment levels mirrored the increased relative plasmid-mediated toxicity of pU6.g1/Cas9 in hematopoietic stem/progenitor cells (HSPCs), highlighting the value for the further improvements of CRISPR/Cas9 delivery in primary human HSPCs.

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.

There is a critical need for increasing the diversity representation among clinicians and researchers in academic medicine, including departments of psychiatry and psychology. Mentorship of under-represented groups has been identified as an important way to remediate diversity-related barriers in the field. This paper outlines the early development and pilot implementation of a diversity mentorship program at Brown University. In an effort to inform and guide future diversity programs, we discuss the challenges faced in creating the program, the successes experienced during the first year, and the future directions undertaken as a means for improving the program.