INTRODUCTION — Gene therapy (GT) for inborn errors of immunity (IEI; previously called primary immunodeficiency disorders [PIDs]) involves restoring a functional copy of the defective gene into the affected patient's own hematopoietic stem cells (HSCs) by gene addition or gene editing. It is one of the two modalities with the potential to cure patients with IEI, the other being hematopoietic cell transplantation (HCT), which provides healthy donor HSCs that will differentiate into mature functional immune cells in an affected patient. HCT for IEI is discussed in detail separately. (See "Hematopoietic cell transplantation for severe combined immunodeficiencies" and "Hematopoietic cell transplantation for non-SCID inborn errors of immunity".)
Traditionally, GT is accomplished by removing HSCs from an affected patient, adding ex vivo a corrected gene copy that integrates into chromosomal deoxyribonucleic acid (DNA; gene addition), and then returning the cells to the patient. Potential alternatives or adjunctive approaches to gene addition therapy or HCT are under development, including gene editing approaches. The use of standard and alternative approaches to GT for IEI are reviewed in this topic and are discussed in general separately. (See "Overview of gene therapy, gene editing, and gene silencing".)
A general overview of IEI management is provided separately, and treatments for specific IEI are discussed in the disease-specific topics. (See "Inborn errors of immunity (primary immunodeficiencies): Overview of management".)
RATIONALE — The majority of hematopoietic cell transplant (HCT) candidates do not have an eligible fully human leukocyte antigen (HLA) matched family donor. Use of less-well-matched donor sources can carry higher risks of graft rejection or graft-versus-host disease and require greater donor graft manipulation or more potent pretransplant immunosuppression in the recipient. An alternative, potentially safer approach is using the patient's own (autologous) hematopoietic stem cells (HSCs) with the disease-related gene corrected to eliminate the risks of graft rejection or graft-versus-host disease. (See "Hematopoietic cell transplantation for severe combined immunodeficiencies", section on 'Donor choice' and "Hematopoietic cell transplantation for non-SCID inborn errors of immunity", section on 'Donor choice'.)
●Overview – Traditional GT (gene addition) uses the unique ability of certain viruses to integrate into the host genome. Modified viruses into which IEI genes have been introduced while removing the ability to self-replicate have made possible ex vivo correction of certain IEI genotypes. Retroviruses including gamma retroviruses (eg, Moloney murine leukemia virus [MMLV]) and lentiviruses (eg, human immunodeficiency virus [HIV]), have been exploited for therapeutic applications where stable, long-term integration of a correct complementary DNA (cDNA) into host hematopoietic stem cells (HSCs) is required. Retroviral vectors have been engineered to infect a wide variety of cell types and have a relatively low toxicity profile compared with other viral vectors such as adenovirus . Lentiviral vectors have an advantage over first-generation retroviruses in that they are able to infect both nondividing and dividing cells, leading to increased transduction efficiency compared with gamma-retroviral vectors, which can only infect dividing cells. (See "Overview of gene therapy, gene editing, and gene silencing", section on 'Gene therapy'.)
The process of gene correction starts with the collection of stem cells from the patient. Autologous bone marrow or peripheral mobilized HSCs are harvested and enriched for HSCs using selective binding to anti-CD34 antibodies. CD34+ cells, which are enriched for HSCs, are then briefly cultured and stimulated with cytokines. The activated CD34+ stem cells are transduced in vitro with a retroviral or lentiviral vector carrying a normal copy of the target gene cDNA, with expression driven by a suitable promoter for the desired cell lineage [2-4]. The retroviral ribonucleic acid (RNA) is reverse transcribed to DNA and integrated into the host genome. Transduced cells undergo sterility and quality checks and upon release are then infused into the patient. New immune cells expressing the transgene are usually first detected in peripheral blood at approximately four to six weeks postinfusion, but it generally takes at least three to six months for functional immune reconstitution to occur.
●Efficacy – Initial studies showed that gamma-retroviral GT was less successful in older children and young adults with X-linked severe combined immunodeficiency (X-SCID), many of whom had previously received haploidentical stem cell transplantation. Four out of five older patients (age 10 to 20 years) who received autologous gene-modified stem cells had poor immune reconstitution after failed allogeneic hematopoietic cell transplantation (HCT) [5-7]. Despite this, increased growth rates and subjective improvements in well-being were reported in two of the five patients . (See "X-linked severe combined immunodeficiency (X-SCID)", section on 'Gene therapy'.)
Two developments, the use of lentiviral vectors that have higher transduction efficiency  and the administration of low-dose preconditioning to open marrow niches, led to effectively restored immune function in older patients with persistent immune dysfunction despite haploidentical stem cell transplantation . The benefit of reduced-intensity preconditioning to open bone marrow niches for incoming corrected stem cells was previously demonstrated in GT for adenosine deaminase deficiency (ADA) SCID, with improved immune reconstitution and development of gene-marked B and natural killer (NK) cells [8,9].
●Safety – Despite improvements in T, NK, and B cell reconstitution, serious adverse events occurred with successful GT with the first-generation gamma retroviruses that used viral long terminal repeat (LTR) sequences to drive expression of the correct cDNA, most notably insertional mutagenesis resulting in lymphoproliferative disease.
In early trials with gamma-retroviral vectors in patients with X-SCID, 6 of 20 participants developed leukemia due to integration of the provirus into or close to proto-oncogenes, cancer-associated genes, and growth-regulating genes. Four of the six children with iatrogenic leukemia were successfully treated with chemotherapy, while one died of his disease [10,11]. In the patients who recovered, polyclonal populations of transduced T cells were restored after chemotherapy for their leukemia. (See "X-linked severe combined immunodeficiency (X-SCID)", section on 'Gene therapy'.)
Increased incidence of leukemia with the use of retroviral vectors is thought to be multifactorial:
•Retroviral vectors preferentially integrate into gene-rich regions, especially near transcription start sites and sometimes into common fragile sites. Two genes that were frequently activated by this mechanism were the LIM domain only 2 (LMO2) and cyclin D2 (CCND2) oncogenes [12,13]. Activation of LMO2 is associated with the development of T cell acute lymphoblastic leukemia . This preferential integration pattern of gamma retroviral vectors, combined with activation of adjacent gene expression by the vector LTR sequences, led to the increased risk of leukemia. Thus, in multiple patients, the therapeutic provirus was shown to have integrated within or near the LMO2 oncogene locus, resulting in aberrant LMO2 expression [12-14].
•In mouse models, the therapeutic gene itself, interleukin 2 receptor subunit gamma (IL2RG), was also subsequently shown to have oncogenic potential when overexpressed . (See "Classification, cytogenetics, and molecular genetics of acute lymphoblastic leukemia/lymphoma", section on 'T-ALL/LBL'.)
Additional clinical trials of retroviral vector GT were associated with an increased incidence of leukemia . Due to this, the US Food and Drug Administration (FDA) placed a clinical hold on all active GT trials using retroviral vectors to insert genes into blood stem cells . GT was allowed to continue on a case-by-case basis if no other treatment was available. Since then, several measures to improve safety, including use of self-inactivating (SIN) vectors or promoters other than LTRs to drive gene expression, are in use in clinical trials for the treatment of X-SCID [2-4,18], ADA-SCID, and Artemis SCID (ART-SCID); X-linked chronic granulomatous disease (X-CGD); and Wiskott-Aldrich syndrome (WAS) and are discussed in greater detail below. (See 'Lymphoproliferative disease with gene addition' below.)
●Opening marrow niches for long-term multilineage engraftment – Successful GT for IEI requires sufficient open marrow niches to allow adequate gene-corrected autologous donor HSCs to engraft and correct the disease manifestation. Alkylating agents such as busulfan and melphalan are the most commonly used preconditioning agents for opening marrow niches. These alkylating agents have the potential for late effects in infants and young children. Thus, nonchemotherapy approaches are under development including monoclonal antibodies targeting the c-Kit proto-oncogene, a stem cell factor receptor with tyrosine kinase activity that is expressed at the surface of HSCs but not more mature blood lineages .
In ADA-SCID, low-dose busulfan prior to GT with a lentiviral vector appears to result in successful multilineage engraftment and both T and B cell reconstitution . Similarly, targeted conditioning with low total dose busulfan in patients with X-SCID has been successful in opening marrow niches and leading to durable gene-corrected donor stem cell engraftment . Finally, X-CGD GT trials demonstrated that use of nonmyeloablative conditioning is essential for long-term presence of adequate numbers of functional granulocytes .
Low-dose conditioning to open marrow niches is probably necessary for other SCID genotypes, such as recombination-activating gene (RAG) SCID and ART-SCID. In the mouse model of ART-SCID, the marrow is more susceptible to alkylating agents than wild-type marrow, so lower doses of chemotherapy may suffice in humans .
Gene editing — Gene editing is an investigational therapy that involves targeted gene correction of a pathogenic variant while maintaining use of the endogenous gene promoter and other regulatory elements. Because the mutated gene is corrected at its endogenous locus and remains under physiologic control in its normal chromosomal context, a therapeutic benefit is expected without the associated complications of insertional mutagenesis, imperfect gene regulation, and transgene silencing sometimes seen with gene addition therapy. Gene editing may be particularly beneficial for defects in genes with closely regulated expression.
Gene editing is based upon homologous recombination. A cDNA cassette that includes the corrective gene segment (or even entire correct cDNA) is targeted to the desired site of integration using flanking "homology arms" as templates for homologous recombination. Specific DNA sequences are targeted and edited using one of several programmable biologic nucleases with specifically tailored recognition sites, including the clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) system, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs) [24,25]. (See "Overview of gene therapy, gene editing, and gene silencing", section on 'Gene editing'.)
Gene editing is under investigation for several forms of SCID, X-linked hyperimmunoglobulin M syndrome (X-HIGM), X-linked agammaglobulinemia (XLA), IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome, leukocyte adhesion deficiency (LAD), and X-CGD, among others . While genome editing platforms still face many of the challenges involved in working with HSCs such as potential genotoxicity and low efficiency, animal models and in vitro studies have demonstrated the ability to correct IEI gene defects. It is likely that further refinement of the technology will lead to a role in clinical treatment.
LIMITATIONS — There are several important obstacles to overcome before GT has more widespread clinical utility .
Lymphoproliferative disease with gene addition — One concern is the association of lymphoproliferative disease with gene addition. (See 'Gene addition' above.)
Several approaches to reduce the risk of insertional oncogenesis have been developed:
●Use of other viral vectors – Lentiviruses, such as human immunodeficiency virus (HIV), are a subtype of retroviruses that are desirable as vectors due to their ability to infect both nondividing and dividing cells. Additionally, lentiviral vectors do not preferentially integrate near enhancers and promoters; therefore, they are less likely to integrate into regions that control expression of proto-oncogenes [10,28,29]. However, lentiviruses still have a tendency to insert into open chromatin, which is where transcribed genes are located, leading to the production of aberrantly spliced transcripts and deregulated (increased or decreased) gene expression [30,31].
●Replication-defective vectors – Replication of retroviruses can lead to infection of other cells and tissues with potential for oncogenesis. Thus, replication is undesirable after integration of the therapeutic gene has occurred. Recoding the vector sequences removes elements associated with replication.
●Self-inactivating (SIN) gamma-retroviral vectors – Gamma-retroviral long terminal repeats (LTRs) that have intrinsic promoter-enhancing activity are inactivated by the introduction of deletions in these sequences [10,18]. SIN gamma-retroviral vectors have decreased mutagenesis and lymphoproliferation .
●Chromatin remodeling elements – The incorporation of chromatin remodeling elements such as insulators shield neighboring genes from influence of sequences within the vector itself .
Other gene addition obstacles — Several other important technical challenges for gene addition include:
●Isolation and culture of the appropriate cell population for transduction. Transduction of pluripotent hematopoietic stem cells (HSCs) is ideal for disorders involving genes expressed in multiple leukocyte lineages . The cell surface marker CD34 is present on HSC as well as early committed hematopoietic progenitor cells. It is possible to isolate significant quantities of CD34+ cells from bone marrow or after mobilization into the peripheral circulation by apheresis. When activated by brief culture with cytokines, CD34+ cells can be readily transduced. Prolonged culture, however, causes these cells to lose their totipotent differentiation potential.
●The efficiency with which genes are transduced into target cells. In early trials, 0.1 percent or fewer blood leukocytes carried retroviral vector markers over the long term in clinical trials . Improving transduction efficiency is an issue if transduced cells do not have a significant survival advantage and/or differentiation advantage in comparison with cells that have not been transduced.
●Correction of defects in which proper regulation of gene expression is required necessitates vectors containing appropriate control sequences or site-specific gene integration.
Gene editing obstacles — Limitations of gene editing include:
●Decrease in rates of gene correction going from in vitro to in vivo assays, suggesting lower efficiency of homology-directed repair in the more primitive HSC population.
●Development of editing approaches for each gene target/disease is labor intensive and, with current methodology, prohibitive for large-scale clinical use.
●Relatively high rates of gene correction are needed to fully cure disease in some conditions (eg, Wiskott-Aldrich syndrome [WAS]).
GENE THERAPY FOR SPECIFIC DISORDERS
Adenosine deaminase deficiency SCID — Adenosine deaminase (ADA) is a critical enzyme that is found in all nucleated cells and detoxifies metabolites that result from the purine metabolism pathway. Loss of ADA activity caused by pathogenic variants in the ADA gene leads to severe combined immunodeficiency (SCID). This autosomal recessive condition causes profoundly diminished T cell, natural killer (NK) cell, and B cell development because elevated concentrations of purine metabolites are preferentially toxic to lymphoid cells. ADA-SCID was the first human disease to be treated with autologous GT. Autologous CD34+ enriched hematopoietic stem cell GT (HSC-GT) for ADA-SCID was approved for clinical use in the European Union in 2016 and has been available in the United States through clinical trials. There is consensus that, where available, HSC-GT is a reasonable first-line treatment choice for patients with ADA deficiency. Outcomes have improved with the use of self-inactivating lentiviral vectors, refined stem cell processing methods, and enhanced engraftment of the autologous gene-corrected stem cells through reduced-intensity conditioning with low-dose, pharmacokinetic-adjusted busulfan (typically one-third of the tissue exposure used in myeloablative protocols for allogeneic transplantation). Treatment of ADA deficiency, including timing of discontinuation of pegylated adenosine deaminase (PEG-ADA) enzyme replacement therapy, is discussed in greater detail separately. (See "Adenosine deaminase deficiency: Treatment and prognosis", section on 'Preferred definitive therapy for ADA-SCID'.)
X-linked SCID — X-linked SCID (X-SCID) is caused by pathogenic variants in the gene encoding the interleukin (IL) 2 receptor gamma chain (IL2RG or the common gamma chain). This receptor subunit is shared by at least six different cytokine receptor complexes: IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Common gamma chain pathogenic variants result in the absence of T cell and NK cell development and impairment of B cell function. Curative therapy is often achieved through HCT. Only patients who lack a human leukocyte antigen (HLA) identical family donor for HCT are considered candidates for GT. Initial GT with gamma-retroviral vectors was successful in reconstituting immune function in infants with X-SCID, but a high rate of insertional mutagenesis was seen. Both infants and older patients with X-SCID have demonstrated immune reconstitution and resolution of infections with GT using SIN vectors. Long-term follow-up is still necessary to confirm lack of leukemogenesis with the second- and third-generation modified vectors. GT for X-SCID is discussed in greater detail separately. (See "X-linked severe combined immunodeficiency (X-SCID)", section on 'Gene therapy'.)
Artemis SCID — Artemis is an exonuclease essential for the repair of DNA double-strand breaks initiated during V(D)J recombination in the process of T and B cell maturation. Pathogenic variants in DNA cross-link repair protein 1C (DCLRE1C), the gene encoding Artemis, cause a form of T-B-NK+ SCID (ART-SCID) and confer sensitivity to ionizing radiation and alkylating chemotherapy. Conditioning regimens are poorly tolerated and often lead to high mortality and/or late complications. However, without alkylating pretransplant conditioning, patients usually have poor engraftment or graft rejection. Thus, autologous GT with only low-dose preconditioning would be beneficial in this population. Initial mouse model and in vitro study results were promising, and clinical trials are underway. GT for ART-SCID is discussed in greater detail separately. (See "T-B-NK+ SCID: Management", section on 'Gene therapy'.)
X-linked chronic granulomatous disease — X-linked chronic granulomatous disease (X-CGD) results from abnormal phagocyte function due to pathogenic variants in the cytochrome b-245 beta chain (CYBB) gene encoding for gp91phox, the redox center of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Chronic catalase-positive organism infection and inflammatory bowel disease are hallmarks of the condition. A limited number of patients with CGD have been treated with GT, and those treated have been high-risk patients with profound oxidase deficiency leading to severe complications and those who lack an HLA-matched donor. Early trials for X-CGD were similar to those that targeted X-SCID and Wiskott-Aldrich syndrome (WAS) in terms of methodologies and vectors used. Initial success rates were low, with severe complications that included death related to abnormal clonal hematopoiesis secondary to vector integration events. Subsequent ongoing clinical trials have used a SIN lentiviral vector with a chimeric promoter and nonmyeloablative conditioning. GT for X-CGD is reviewed in greater detail separately. (See "Chronic granulomatous disease: Treatment and prognosis", section on 'Gene therapy/gene repair'.)
Wiskott-Aldrich syndrome — WAS is an X-linked disorder caused by loss of function of the WAS gene. The condition affects the immunohematopoietic system and results in microthrombocytopenia and lymphoid and myeloid cell dysfunction. Infections, eczema, bleeding, and autoimmunity are hallmarks of this condition. The first GT trials with gamma-retroviral vector resulted in significant clinical benefit but were associated with high rates of insertional mutagenesis and resultant late-onset hematologic malignancy in most patients. Lentiviral-based GT trials restricted to patients without a fully matched donor were subsequently initiated, with encouraging results in both improvement in thrombocytopenia, susceptibility to infections, and eczema and no evidence of lymphoproliferative disease. GT for WAS is discussed in greater detail separately. (See "Wiskott-Aldrich syndrome", section on 'Gene therapy'.)
Other inborn errors of immunity — Gene addition therapies and early clinical trials are under development for several other IEI including SCID due to recombination-activating genes (RAG) 1 and 2 deficiency, autosomal-recessive forms of CGD (p47 and p67), and leukocyte adhesion deficiency (LAD).
Other IEI that are caused by genes involved in processes that require precise temporal and physiologic control, such as cell activation and intracellular signaling, may be more amenable to gene editing than gene addition therapy. As such, preclinical investigation of gene editing is underway for X-linked hyper-IgM syndrome (X-HIGM) X-linked agammaglobulinemia (XLA), and IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome.
SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Inborn errors of immunity (previously called primary immunodeficiencies)".)
SUMMARY AND RECOMMENDATIONS
●Overview – Gene therapy (GT) for inborn errors of immunity (IEI; previously called primary immunodeficiency disorders [PIDs]) involves restoring a functional copy of the defective gene into the affected patient's own hematopoietic stem cells (HSCs) by gene addition or gene editing. It is one of the two modalities with the potential to cure patients with IEI, the other being hematopoietic cell transplantation (HCT). (See 'Introduction' above and 'Rationale' above.)
●Gene addition – Traditional GT (gene addition) uses the unique ability of certain viruses (eg, retroviruses, lentiviruses) to integrate into the host genome. Modified viruses into which IEI genes have been introduced while removing the ability to self-replicate are used to integrate a corrected segment of deoxyribonucleic acid (DNA) into host HSCs. New immune cells expressing the transgene are usually first detected in peripheral blood at approximately four to six weeks postinfusion, but it generally takes at least three to six months for functional immune reconstitution to occur. Efficacy has improved with the use of lentiviral vectors that have higher transduction efficiency and the administration of low-dose preconditioning to open marrow niches for incoming corrected stem cells. (See 'Gene addition' above.)
●Gene editing – Gene editing is an investigational therapy that involves targeted gene correction of a pathogenic variant at its endogenous locus, maintaining use of the endogenous gene promoter and other regulatory elements. The corrected gene remains under physiologic control in its normal chromosomal context. Thus, a therapeutic benefit is expected without the associated complications of insertional mutagenesis, imperfect gene regulation, and transgene silencing sometimes seen with gene addition therapy. Gene editing may be particularly beneficial for defects in genes with closely regulated expression. (See 'Gene editing' above.)
●Lymphoproliferative disease – Early GT trials for IEI that used retroviral vectors were associated with insertional mutagenesis and hematologic malignancy, except for adenosine deaminase deficiency severe combined immunodeficiency (ADA-SCID). Several approaches to decrease this risk include use of other viral vectors (eg, lentiviruses, foamy viruses), replication-defective vectors, self-inactivating (SIN) gamma-retroviral vectors, and chromatin remodeling elements. (See 'Gene addition' above and 'Lymphoproliferative disease with gene addition' above.)
●GT for specific IEI – Successful reconstitution of immune function using lentiviral vector GT has been demonstrate for ADA-SCID, X-linked SCID (X-SCID), X-linked chronic granulomatous disease (X-CGD), and Wiskott-Aldrich syndrome (WAS), with no vector-related complications reported. (See 'Gene therapy for specific disorders' above.)
ACKNOWLEDGMENT — The UpToDate editorial staff acknowledges Francisco A Bonilla, MD, PhD, and E Richard Stiehm, MD, who contributed to earlier versions of this topic review as author and Section Editor, respectively.
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