Anti-CD45 PBD-based antibody-drug conjugates are effective targeted conditioning agents for gene therapy and stem cell transplant.

Stem cell gene therapy and hematopoietic stem cell transplantation (SCT) require conditioning to ablate the recipient's hematopoietic stem cells (HSCs) and create a niche for gene-corrected/donor HSCs. Conventional conditioning agents are non-specific, leading to off-target toxicities and resulting in significant morbidity and mortality. We developed tissue-specific anti-human CD45 antibody-drug conjugates (ADCs), using rat IgG2b anti-human CD45 antibody clones YTH24.5 and YTH54.12, conjugated to cytotoxic pyrrolobenzodiazepine (PBD) dimer payloads with cleavable (SG3249) or non-cleavable (SG3376) linkers. In vitro, these ADCs internalized to lysosomes for drug release, resulting in potent and specific killing of human CD45+ cells. In humanized NSG mice, the ADCs completely ablated human HSCs without toxicity to non-hematopoietic tissues, enabling successful engraftment of gene-modified autologous and allogeneic human HSCs. The ADCs also delayed leukemia onset and improved survival in CD45+ tumor models. These data provide proof of concept that conditioning with anti-human CD45-PBD ADCs allows engraftment of donor/gene-corrected HSCs with minimal toxicity to non-hematopoietic tissues. Our anti-CD45-PBDs or similar agents could potentially shift the paradigm in transplantation medicine that intensive chemo/radiotherapy is required for HSC engraftment after gene therapy and allogeneic SCT. Targeted conditioning both improve the safety and minimize late effects of these procedures, which would greatly increase their applicability.

Targeting the central nervous system in lysosomal storage diseases: Strategies to deliver therapeutics across the blood-brain barrier.

Lysosomal storage diseases (LSDs) are multisystem inherited metabolic disorders caused by dysfunctional lysosomal activity, resulting in the accumulation of undegraded macromolecules in a variety of organs/tissues, including the central nervous system (CNS). Treatments include enzyme replacement therapy, stem/progenitor cell transplantation, and in vivo gene therapy. However, these treatments are not fully effective in treating the CNS as neither enzymes, stem cells, nor viral vectors efficiently cross the blood-brain barrier. Here, we review the latest advancements in improving delivery of different therapeutic agents to the CNS and comment upon outstanding questions in the field of neurological LSDs.

The transformative potential of HSC gene therapy as a genetic medicine.

Hematopoietic stem cells (HSCs) are precursor cells that give rise to blood, immune and tissue-resident progeny in humans. Their position at the starting point of hematopoiesis offers a unique therapeutic opportunity to treat certain hematologic diseases by implementing corrective changes that are subsequently directed through to multiple cell lineages. Attempts to exploit HSCs clinically have evolved over recent decades, from initial approaches that focused on transplantation of healthy donor allogeneic HSCs to treat rare inherited monogenic hematologic disorders, to more contemporary genetic modification of autologous HSCs offering the promise of benefits to a wider range of diseases. We are on the cusp of an exciting new era as the transformative potential of HSC gene therapy to offer durable delivery of gene-corrected cells to a range of tissues and organs, including the central nervous system, is beginning to be realized. This article reviews the rationale for targeting HSCs, the approaches that have been used to date for delivering therapeutic genes to these cells, and the latest technological breakthroughs in manufacturing and vector design. The challenges faced by the biotechnology cell and gene therapy sector in the commercialization of HSC gene therapy are also discussed.

Lentiviral Gene Transfer Corrects Immune Abnormalities in XIAP Deficiency.

BACKGROUND: X-linked inhibitor of apoptosis protein (XIAP) deficiency is a severe immunodeficiency with clinical features including hemophagocytic lymphohistiocytosis (HLH) and inflammatory bowel disease (IBD) due to defective NOD2 responses. Management includes immunomodulatory therapies and hematopoietic stem cell transplant (HSCT). However, this cohort is particularly susceptible to the chemotherapeutic regimens and acutely affected by graft-vs-host disease (GvHD), driving poor long-term survival in transplanted patients. Autologous HSC gene therapy could offer an alternative treatment option and would abrogate the risks of alloreactivity. METHODS: Hematopoietic progenitor (Lin-ve) cells from XIAPy/- mice were transduced with a lentiviral vector encoding human XIAP cDNA before transplantation into irradiated XIAP y/- recipients. After 12 weeks animals were challenged with the dectin-1 ligand curdlan and recovery of innate immune function was evaluated though analysis of inflammatory cytokines, body weight, and splenomegaly. XIAP patient-derived CD14+ monocytes were transduced with the same vector and functional recovery was demonstrated using in vitro L18-MDP/NOD2 assays. RESULTS: In treated XIAPy/- mice, ~40% engraftment of gene-corrected Lin-ve cells led to significant recovery of weight loss, splenomegaly, and inflammatory cytokine responses to curdlan, comparable to wild-type mice. Serum IL-6, IL-10, MCP-1, and TNF were significantly reduced 2-h post-curdlan administration in non-corrected XIAPy/- mice compared to wild-type and gene-corrected animals. Appropriate reduction of inflammatory responses was observed in gene-corrected mice, whereas non-corrected mice developed an inflammatory profile 9 days post-curdlan challenge. In gene-corrected patient CD14+ monocytes, TNF responses were restored following NOD2 activation with L18-MDP. CONCLUSION: Gene correction of HSCs recovers XIAP-dependent immune defects and could offer a treatment option for patients with XIAP deficiency.

Long-Term Immune Recovery After Hematopoietic Stem Cell Transplantation for ADA Deficiency: a Single-Center Experience.

Unconditioned hematopoietic stem cell transplantation (HSCT) is the recommended treatment for patients with adenosine deaminase (ADA)-deficient severe combined immunodeficiency with an HLA-matched sibling donor (MSD) or family donor (MFD). Improved overall survival (OS) has been reported compared to the use of unrelated donors, and previous studies have demonstrated that adequate cellular and humoral immune recovery can be achieved even in the absence of conditioning. Detailed insight of the long-term outcome is still limited. We aim to address this by studying a large single-center cohort of 28 adenosine deaminase-deficient patients who underwent a total of 31 HSCT procedures, of which more than half were unconditioned. We report an OS of 85.7% and event-free survival of 71% for the entire cohort, with no statistically significant differences after procedures using related or unrelated HLA-matched donors. We find that donor engraftment in the myeloid compartment is significantly diminished in unconditioned procedures, which typically use a MSD or MFD. This is associated with poor metabolic correction and more frequent failure to discontinue immunoglobulin replacement therapy. Approximately one in four patients receiving an unconditioned procedure required a second procedure, whereas the use of reduced intensity conditioning (RIC) prior to allogeneic transplantation improves the long-term outcome by achieving better myeloid engraftment, humoral immune recovery, and metabolic correction. Further longitudinal studies are needed to optimize future management and guidelines, but our findings support a potential role for the routine use of RIC in most ADA-deficient patients receiving an HLA-identical hematopoietic stem cell transplant, even when a MSD or MFD is available.

Retrospective, Landmark Analysis of Long-term Adult Morbidity Following Allogeneic HSCT for Inborn Errors of Immunity in Infancy and Childhood.

PURPOSE: Allogeneic hematopoietic stem cell transplant (HSCT) remains the treatment of choice for patients with inborn errors of immunity (IEI). There is little published medical outcome data assessing late medical complications following transition to adult care. We sought to document event-free survival (EFS) in transplanted IEI patients reaching adulthood and describe common late-onset medical complications and factors influencing EFS. METHODS: In this landmark analysis, 83 adults surviving 5 years or more following prior HSCT in childhood for IEI were recruited. The primary endpoint was event-free survival, defined as time post-first HSCT to graft failure, graft rejection, chronic infection, life-threatening or recurrent infections, malignancy, significant autoimmune disease, moderate to severe GVHD or major organ dysfunction. All events occurring less than 5 years post-HSCT were excluded. RESULTS: EFS was 51% for the whole cohort at a median of 20 years post HSCT. Multivariable analysis identified age at transplant and whole blood chimerism as independent predictors of long-term EFS. Year of HSCT, donor, conditioning intensity and underlying diagnosis had no significant impact on EFS. 59 events occurring beyond 5 years post-HSCT were documented in 37 patients (45% cohort). A total of 25 patients (30% cohort) experienced ongoing significant complications requiring active medical intervention at last follow-up. CONCLUSION: Although most patients achieved excellent, durable immune reconstitution with infrequent transplant-related complications, very late complications are common and associated with mixed chimerism post-HSCT. Early intervention to correct mixed chimerism may improve long-term outcomes and adult health following HSCT for IEI in childhood.

Clinical and immunological features in a cohort of patients with partial DiGeorge syndrome followed at a single center.

DiGeorge syndrome (DGS) is a primary immunodeficiency characterized by various degrees of T-cell deficiency. In partial DGS (pDGS), other risk factors could predispose to recurrent infections, autoimmunity, and allergy. The aim of this study was to assess the effect of different factors in the development of infections, autoimmunity, and/or allergy in patients with pDGS. We studied 467 pDGS patients in follow-up at Great Ormond Street Hospital. Using a multivariate approach, we observed that palatal anomalies represent a risk factor for the development of recurrent otitis media with effusion. Gastroesophageal reflux/dysphagia and asthma/rhinitis represent a risk factor for the development of recurrent upper respiratory tract infections. Allergy and autoimmunity were associated with persistently low immunoglobulin M levels and lymphopenia, respectively. Patients with autoimmunity showed lower levels of CD3+, CD3+CD4+, and naïve CD4+CD45RA+CD27+ T lymphocytes compared with pDGS patients without autoimmunity. We also observed that the physiological age-related decline of the T-cell number was slower in pDGS patients compared with age-matched controls. The age-related recovery of the T-cell number depended on a homeostatic peripheral proliferation of T cells, as suggested by an accelerated decline of the naïve T lymphocytes in pDGS as well as a more skewed T-cell repertoire in older pDGS patients. These evidences suggest that premature CD4+ T-cell aging and lymphopenia induced spontaneous peripheral T-cell proliferation might contribute to the pathogenesis of autoimmunity in patients with pDGS. Infections in these patients represent, in most of the cases, a complication of anatomical or gastroenterological anomalies rather than a feature of the underlying immunodeficiency.

Busulfan Pharmacokinetics in Adenosine Deaminase-Deficient Severe Combined Immunodeficiency Gene Therapy.

The pharmacokinetics of low-dose busulfan (BU) were investigated as a nonmyeloablative conditioning regimen for autologous gene therapy (GT) in pediatric subjects with adenosine deaminase-deficient severe combined immunodeficiency disease (ADA SCID). In 3 successive clinical trials, which included either γ-retroviral (γ-RV) or lentiviral (LV) vectors, subjects were conditioned with BU using different dosing nomograms. The first cohort received BU doses based on body surface area (BSA), the second cohort received doses based on actual body weight (ABW), and in the third cohort, therapeutic drug monitoring (TDM) was used to target a specific area under the concentration-time curve (AUC). Neither BSA-based nor ABW-based dosing achieved a consistent cumulative BU AUC; in contrast, TDM-based dosing led to more consistent AUC. BU clearance increased as subject age increased from birth to 18 months. However, weight and age alone were insufficient to accurately predict the dose that would consistently achieve a target AUC. Furthermore, various clinical, laboratory, and genetic factors (eg, genotypes for glutathione-S-transferase isozymes known to participate in BU metabolism) were analyzed, but no single finding predicted subjects with rapid versus slow clearance. Analysis of BU AUC and the postengraftment vector copy number (VCN) in granulocytes, a surrogate marker of the level of engrafted gene-modified hematopoietic stem and progenitor cells (HSPCs), demonstrated gene marking at levels sufficient for therapeutic benefit in the subjects who had achieved the target BU AUC. Although many factors determine the ultimate engraftment following GT, this work demonstrates that the BU AUC correlated with the eventual level of engrafted gene-modified HSPCs within a vector group (γ-RV versus LV), with significantly higher levels of granulocyte VCN in the recipients of LV-modified grafts compared to recipients of γ-RV-transduced grafts. Taken together, these findings provide insight into low-dose BU pharmacokinetics in the unique setting of autologous GT for ADA SCID, and these dosing principles may be applied to future GT trials using low-dose BU to open the bone marrow niche.

Hematopoietic Stem-Cell Gene Therapy for Cerebral Adrenoleukodystrophy.

BACKGROUND: In X-linked adrenoleukodystrophy, mutations in ABCD1 lead to loss of function of the ALD protein. Cerebral adrenoleukodystrophy is characterized by demyelination and neurodegeneration. Disease progression, which leads to loss of neurologic function and death, can be halted only with allogeneic hematopoietic stem-cell transplantation. METHODS: We enrolled boys with cerebral adrenoleukodystrophy in a single-group, open-label, phase 2-3 safety and efficacy study. Patients were required to have early-stage disease and gadolinium enhancement on magnetic resonance imaging (MRI) at screening. The investigational therapy involved infusion of autologous CD34+ cells transduced with the elivaldogene tavalentivec (Lenti-D) lentiviral vector. In this interim analysis, patients were assessed for the occurrence of graft-versus-host disease, death, and major functional disabilities, as well as changes in neurologic function and in the extent of lesions on MRI. The primary end point was being alive and having no major functional disability at 24 months after infusion. RESULTS: A total of 17 boys received Lenti-D gene therapy. At the time of the interim analysis, the median follow-up was 29.4 months (range, 21.6 to 42.0). All the patients had gene-marked cells after engraftment, with no evidence of preferential integration near known oncogenes or clonal outgrowth. Measurable ALD protein was observed in all the patients. No treatment-related death or graft-versus-host disease had been reported; 15 of the 17 patients (88%) were alive and free of major functional disability, with minimal clinical symptoms. One patient, who had had rapid neurologic deterioration, had died from disease progression. Another patient, who had had evidence of disease progression on MRI, had withdrawn from the study to undergo allogeneic stem-cell transplantation and later died from transplantation-related complications. CONCLUSIONS: Early results of this study suggest that Lenti-D gene therapy may be a safe and effective alternative to allogeneic stem-cell transplantation in boys with early-stage cerebral adrenoleukodystrophy. Additional follow-up is needed to fully assess the duration of response and long-term safety. (Funded by Bluebird Bio and others; STARBEAM ClinicalTrials.gov number, NCT01896102 ; ClinicalTrialsRegister.eu number, 2011-001953-10 .).

Consensus approach for the management of severe combined immune deficiency caused by adenosine deaminase deficiency.

Inherited defects in adenosine deaminase (ADA) cause a subtype of severe combined immunodeficiency (SCID) known as severe combined immune deficiency caused by adenosine deaminase defects (ADA-SCID). Most affected infants can receive a diagnosis while still asymptomatic by using an SCID newborn screening test, allowing early initiation of therapy. We review the evidence currently available and propose a consensus management strategy. In addition to treatment of the immune deficiency seen in patients with ADA-SCID, patients should be followed for specific noninfectious respiratory, neurological, and biochemical complications associated with ADA deficiency. All patients should initially receive enzyme replacement therapy (ERT), followed by definitive treatment with either of 2 equal first-line options. If an HLA-matched sibling donor or HLA-matched family donor is available, allogeneic hematopoietic stem cell transplantation (HSCT) should be pursued. The excellent safety and efficacy observed in more than 100 patients with ADA-SCID who received gammaretrovirus- or lentivirus-mediated autologous hematopoietic stem cell gene therapy (HSC-GT) since 2000 now positions HSC-GT as an equal alternative. If HLA-matched sibling donor/HLA-matched family donor HSCT or HSC-GT are not available or have failed, ERT can be continued or reinstituted, and HSCT with alternative donors should be considered. The outcomes of novel HSCT, ERT, and HSC-GT strategies should be evaluated prospectively in "real-life" conditions to further inform these management guidelines.

Gene therapy for Wiskott-Aldrich syndrome in a severely affected adult.

Until recently, hematopoietic stem cell transplantation was the only curative option for Wiskott-Aldrich syndrome (WAS). The first attempts at gene therapy for WAS using a ϒ-retroviral vector improved immunological parameters substantially but were complicated by acute leukemia as a result of insertional mutagenesis in a high proportion of patients. More recently, treatment of children with a state-of-the-art self-inactivating lentiviral vector (LV-w1.6 WASp) has resulted in significant clinical benefit without inducing selection of clones harboring integrations near oncogenes. Here, we describe a case of a presplenectomized 30-year-old patient with severe WAS manifesting as cutaneous vasculitis, inflammatory arthropathy, intermittent polyclonal lymphoproliferation, and significant chronic kidney disease and requiring long-term immunosuppressive treatment. Following reduced-intensity conditioning, there was rapid engraftment and expansion of a polyclonal pool of transgene-positive functional T cells and sustained gene marking in myeloid and B-cell lineages up to 20 months of observation. The patient was able to discontinue immunosuppression and exogenous immunoglobulin support, with improvement in vasculitic disease and proinflammatory markers. Autologous gene therapy using a lentiviral vector is a viable strategy for adult WAS patients with severe chronic disease complications and for whom an allogeneic procedure could present an unacceptable risk. This trial was registered at www.clinicaltrials.gov as #NCT01347242.

Transfer of gene-corrected T cells corrects humoral and cytotoxic defects in patients with X-linked lymphoproliferative disease.

BACKGROUND: X-linked lymphoproliferative disease 1 arises from mutations in the SH2D1A gene encoding SLAM-associated protein (SAP), an adaptor protein expressed in T, natural killer (NK), and NKT cells. Defects lead to abnormalities of T-cell and NK cell cytotoxicity and T cell-dependent humoral function. Clinical manifestations include hemophagocytic lymphohistiocytosis, lymphoma, and dysgammaglobulinemia. Curative treatment is limited to hematopoietic stem cell transplantation, with outcomes reliant on a good donor match. OBJECTIVES: Because most symptoms arise from defective T-cell function, we investigated whether transfer of SAP gene-corrected T cells could reconstitute known effector cell defects. METHODS: CD3+ lymphocytes from Sap-deficient mice were transduced with a gammaretroviral vector encoding human SAP cDNA before transfer into sublethally irradiated Sap-deficient recipients. After immunization with the T-dependent antigen 4-hydroxy-3-nitrophenylacetly chicken gammaglobulin (NP-CGG), recovery of humoral function was evaluated through germinal center formation and antigen-specific responses. To efficiently transduce CD3+ cells from patients, we generated an equivalent lentiviral SAP vector. Functional recovery was demonstrated by using in vitro cytotoxicity and T follicular helper cell function assays alongside tumor clearance in an in vivo lymphoblastoid cell line lymphoma xenograft model. RESULTS: In Sap-deficient mice 20% to 40% engraftment of gene-modified T cells led to significant recovery of germinal center formation and NP-specific antibody responses. Gene-corrected T cells from patients demonstrated improved cytotoxicity and T follicular helper cell function in vitro. Adoptive transfer of gene-corrected cytotoxic T lymphocytes from patients reduced tumor burden to a level comparable with that seen in healthy donor cytotoxic T lymphocytes in an in vivo lymphoma model. CONCLUSIONS: These data demonstrate that autologous T-cell gene therapy corrects SAP-dependent defects and might offer an alternative therapeutic option for patients with X-linked lymphoproliferative disease 1.

T-cell gene therapy for perforin deficiency corrects cytotoxicity defects and prevents hemophagocytic lymphohistiocytosis manifestations.

BACKGROUND: Mutations in the perforin 1 (PRF1) gene account for up to 58% of familial hemophagocytic lymphohistiocytosis syndromes. The resulting defects in effector cell cytotoxicity lead to hypercytokinemia and hyperactivation with inflammation in various organs. OBJECTIVE: We sought to determine whether autologous gene-corrected T cells can restore cytotoxic function, reduce disease activity, and prevent hemophagocytic lymphohistiocytosis (HLH) symptoms in in vivo models. METHODS: We developed a gammaretroviral vector to transduce murine CD8 T cells in the Prf-/- mouse model. To verify functional correction of Prf-/- CD8 T cells in vivo, we used a lymphocytic choriomeningitis virus (LCMV) epitope-transfected murine lung carcinoma cell tumor model. Furthermore, we challenged gene-corrected and uncorrected mice with LCMV. One patient sample was transduced with a PRF1-encoding lentiviral vector to study restoration of cytotoxicity in human cells. RESULTS: We demonstrated efficient engraftment and functional reconstitution of cytotoxicity after intravenous administration of gene-corrected Prf-/- CD8 T cells into Prf-/- mice. In the tumor model infusion of Prf-/- gene-corrected CD8 T cells eliminated the tumor as efficiently as transplantation of wild-type CD8 T cells. Similarly, mice reconstituted with gene-corrected Prf-/- CD8 T cells displayed complete protection from the HLH phenotype after infection with LCMV. Patients' cells showed correction of cytotoxicity in human CD8 T cells after transduction. CONCLUSION: These data demonstrate the potential application of T-cell gene therapy in reconstituting cytotoxic function and protection against HLH in the setting of perforin deficiency.

Outcome of hematopoietic cell transplantation for DNA double-strand break repair disorders.

BACKGROUND: Rare DNA breakage repair disorders predispose to infection and lymphoreticular malignancies. Hematopoietic cell transplantation (HCT) is curative, but coadministered chemotherapy or radiotherapy is damaging because of systemic radiosensitivity. We collected HCT outcome data for Nijmegen breakage syndrome, DNA ligase IV deficiency, Cernunnos-XRCC4-like factor (Cernunnos-XLF) deficiency, and ataxia-telangiectasia (AT). METHODS: Data from 38 centers worldwide, including indication, donor, conditioning regimen, graft-versus-host disease, and outcome, were analyzed. Conditioning was classified as myeloablative conditioning (MAC) if it contained radiotherapy or alkylators and reduced-intensity conditioning (RIC) if no alkylators and/or 150 mg/m2 fludarabine or less and 40 mg/kg cyclophosphamide or less were used. RESULTS: Fifty-five new, 14 updated, and 18 previously published patients were analyzed. Median age at HCT was 48 months (range, 1.5-552 months). Twenty-nine patients underwent transplantation for infection, 21 had malignancy, 13 had bone marrow failure, 13 received pre-emptive transplantation, 5 had multiple indications, and 6 had no information. Twenty-two received MAC, 59 received RIC, and 4 were infused; information was unavailable for 2 patients. Seventy-three of 77 patients with DNA ligase IV deficiency, Cernunnos-XLF deficiency, or Nijmegen breakage syndrome received conditioning. Survival was 53 (69%) of 77 and was worse for those receiving MAC than for those receiving RIC (P = .006). Most deaths occurred early after transplantation, suggesting poor tolerance of conditioning. Survival in patients with AT was 25%. Forty-one (49%) of 83 patients experienced acute GvHD, which was less frequent in those receiving RIC compared with those receiving MAC (26/56 [46%] vs 12/21 [57%], P = .45). Median follow-up was 35 months (range, 2-168 months). No secondary malignancies were reported during 15 years of follow-up. Growth and developmental delay remained after HCT; immune-mediated complications resolved. CONCLUSION: RIC HCT resolves DNA repair disorder-associated immunodeficiency. Long-term follow-up is required for secondary malignancy surveillance. Routine HCT for AT is not recommended.

Lentiviral Vectors with Cellular Promoters Correct Anemia and Lethal Bone Marrow Failure in a Mouse Model for Diamond-Blackfan Anemia.

Diamond-Blackfan anemia is a congenital erythroid hypoplasia and is associated with physical malformations and a predisposition to cancer. Twenty-five percent of patients with Diamond-Blackfan anemia have mutations in a gene encoding ribosomal protein S19 (RPS19). Through overexpression of RPS19 using a lentiviral vector with the spleen focus-forming virus promoter, we demonstrated that the Diamond-Blackfan anemia phenotype can be successfully treated in Rps19-deficient mice. In our present study, we assessed the efficacy of a clinically relevant promoter, the human elongation factor 1α short promoter, with or without the locus control region of the ß-globin gene for treatment of RPS19-deficient Diamond-Blackfan anemia. The findings demonstrate that these vectors rescue the proliferation defect and improve erythroid development of transduced RPS19-deficient bone marrow cells. Remarkably, bone marrow failure and severe anemia in Rps19-deficient mice was cured with enforced expression of RPS19 driven by the elongation factor 1α short promoter. We also demonstrate that RPS19-deficient bone marrow cells can be transduced and these cells have the capacity to repopulate bone marrow in long-term reconstituted mice. Our results collectively demonstrate the feasibility to cure RPS19-deficient Diamond-Blackfan anemia using lentiviral vectors with cellular promoters that possess a reduced risk of insertional mutagenesis.

Mutations in linker for activation of T cells (LAT) lead to a novel form of severe combined immunodeficiency.

BACKGROUND: Signaling through the T-cell receptor (TCR) is critical for T-cell development and function. Linker for activation of T cells (LAT) is a transmembrane adaptor signaling molecule that is part of the TCR complex and essential for T-cell development, as demonstrated by LAT-deficient mice, which show a complete lack of peripheral T cells. OBJECTIVE: We describe a pedigree affected by a severe combined immunodeficiency phenotype with absent T cells and normal B-cell and natural killer cell numbers. A novel homozygous frameshift mutation in the gene encoding for LAT was identified in this kindred. METHODS: Genetic, molecular, and functional analyses were used to identify and characterize the LAT defect. Clinical and immunologic analysis of patients was also performed and reported. RESULTS: Homozygosity mapping was used to identify potential defective genes. Sanger sequencing of the LAT gene showed a mutation that resulted in a premature stop codon and protein truncation leading to complete loss of function and loss of expression of LAT in the affected family members. We also demonstrate loss of LAT expression and lack of TCR signaling restoration in LAT-deficient cell lines reconstituted with a synthetic LAT gene bearing this severe combined immunodeficiency mutation. CONCLUSION: For the first time, the results of this study show that inherited LAT deficiency should be considered in patients with combined immunodeficiency with T-cell abnormalities.

Thymus transplantation for complete DiGeorge syndrome: European experience.

BACKGROUND: Thymus transplantation is a promising strategy for the treatment of athymic complete DiGeorge syndrome (cDGS). METHODS: Twelve patients with cDGS underwent transplantation with allogeneic cultured thymus. OBJECTIVE: We sought to confirm and extend the results previously obtained in a single center. RESULTS: Two patients died of pre-existing viral infections without having thymopoiesis, and 1 late death occurred from autoimmune thrombocytopenia. One infant had septic shock shortly after transplantation, resulting in graft loss and the need for a second transplant. Evidence of thymopoiesis developed from 5 to 6 months after transplantation in 10 patients. Median circulating naive CD4 counts were 44 × 106/L (range, 11-440 × 106/L) and 200 × 106/L (range, 5-310 × 106/L) at 12 and 24 months after transplantation and T-cell receptor excision circles were 2,238/106 T cells (range, 320-8,807/106 T cells) and 4,184/106 T cells (range, 1,582-24,596/106 T cells). Counts did not usually reach normal levels for age, but patients were able to clear pre-existing infections and those acquired later. At a median of 49 months (range, 22-80 months), 8 have ceased prophylactic antimicrobials, and 5 have ceased immunoglobulin replacement. Histologic confirmation of thymopoiesis was seen in 7 of 11 patients undergoing biopsy of transplanted tissue, including 5 showing full maturation through to the terminal stage of Hassall body formation. Autoimmune regulator expression was also demonstrated. Autoimmune complications were seen in 7 of 12 patients. In 2 patients early transient autoimmune hemolysis settled after treatment and did not recur. The other 5 experienced ongoing autoimmune problems, including thyroiditis (3), hemolysis (1), thrombocytopenia (4), and neutropenia (1). CONCLUSIONS: This study confirms the previous reports that thymus transplantation can reconstitute T cells in patients with cDGS but with frequent autoimmune complications in survivors.

A prospective study on the natural history of patients with profound combined immunodeficiency: An interim analysis.

BACKGROUND: Absent T-cell immunity resulting in life-threatening infections provides a clear rationale for hematopoetic stem cell transplantation (HSCT) in patients with severe combined immunodeficiency (SCID). Combined immunodeficiencies (CIDs) and "atypical" SCID show reduced, not absent T-cell immunity. If associated with infections or autoimmunity, they represent profound combined immunodeficiency (P-CID), for which outcome data are insufficient for unambiguous early transplant decisions. OBJECTIVES: We sought to compare natural histories of severity-matched patients with/without subsequent transplantation and to determine whether immunologic and/or clinical parameters may be predictive for outcome. METHODS: In this prospective and retrospective observational study, we recruited nontransplanted patients with P-CID aged 1 to 16 years to compare natural histories of severity-matched patients with/without subsequent transplantation and to determine whether immunologic and/or clinical parameters may be predictive for outcome. RESULTS: A total of 51 patients were recruited (median age, 9.6 years). Thirteen of 51 had a genetic diagnosis of "atypical" SCID and 14 of 51 of CID. About half of the patients had less than 10% naive T cells, reduced/absent T-cell proliferation, and at least 1 significant clinical event/year, demonstrating their profound immunodeficiency. Nineteen patients (37%) underwent transplantation within 1 year of enrolment, and 5 of 51 patients died. Analysis of the HSCT decisions revealed the anticipated heterogeneity, favoring an ongoing prospective matched-pair analysis of patients with similar disease severity with or without transplantation. Importantly, so far neither the genetic diagnosis nor basic measurements of T-cell immunity were good predictors of disease evolution. CONCLUSIONS: The P-CID study for the first time characterizes a group of patients with nontypical SCID T-cell deficiencies from a therapeutic perspective. Because genetic and basic T-cell parameters provide limited guidance, prospective data from this study will be a helpful resource for guiding the difficult HSCT decisions in patients with P-CID.

How We Manage Adenosine Deaminase-Deficient Severe Combined Immune Deficiency (ADA SCID).

Adenosine deaminase-deficient severe combined immune deficiency (ADA SCID) accounts for 10-15% of cases of human SCID. From what was once a uniformly fatal disease, the prognosis for infants with ADA SCID has improved greatly based on the development of multiple therapeutic options, coupled with more frequent early diagnosis due to implementation of newborn screening for SCID. We review the various treatment approaches for ADA SCID including allogeneic hematopoietic stem cell transplantation (HSCT) from a human leukocyte antigen-matched sibling or family member or from a matched unrelated donor or a haplo-identical donor, autologous HSCT with gene correction of the hematopoietic stem cells (gene therapy-GT), and enzyme replacement therapy (ERT) with polyethylene glycol-conjugated adenosine deaminase. Based on growing evidence of safety and efficacy from GT, we propose a treatment algorithm for patients with ADA SCID that recommends HSCT from a matched family donor, when available, as a first choice, followed by GT as the next option, with allogeneic HSCT from an unrelated or haplo-identical donor or long-term ERT as other options.

A modified γ-retrovirus vector for X-linked severe combined immunodeficiency.

BACKGROUND: In previous clinical trials involving children with X-linked severe combined immunodeficiency (SCID-X1), a Moloney murine leukemia virus-based γ-retrovirus vector expressing interleukin-2 receptor γ-chain (γc) complementary DNA successfully restored immunity in most patients but resulted in vector-induced leukemia through enhancer-mediated mutagenesis in 25% of patients. We assessed the efficacy and safety of a self-inactivating retrovirus for the treatment of SCID-X1. METHODS: We enrolled nine boys with SCID-X1 in parallel trials in Europe and the United States to evaluate treatment with a self-inactivating (SIN) γ-retrovirus vector containing deletions in viral enhancer sequences expressing γc (SIN-γc). RESULTS: All patients received bone marrow-derived CD34+ cells transduced with the SIN-γc vector, without preparative conditioning. After 12.1 to 38.7 months of follow-up, eight of the nine children were still alive. One patient died from an overwhelming adenoviral infection before reconstitution with genetically modified T cells. Of the remaining eight patients, seven had recovery of peripheral-blood T cells that were functional and led to resolution of infections. The patients remained healthy thereafter. The kinetics of CD3+ T-cell recovery was not significantly different from that observed in previous trials. Assessment of insertion sites in peripheral blood from patients in the current trial as compared with those in previous trials revealed significantly less clustering of insertion sites within LMO2, MECOM, and other lymphoid proto-oncogenes in our patients. CONCLUSIONS: This modified γ-retrovirus vector was found to retain efficacy in the treatment of SCID-X1. The long-term effect of this therapy on leukemogenesis remains unknown. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01410019, NCT01175239, and NCT01129544.).