Biomedical subjects

Harry L Malech

Publications and source records attributed to Harry L Malech.

At least 19 recordsLinked to original sources

PubMed2025

Prime Editing for p47phox-Deficient Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is a severe monogenic immunodeficiency caused by damaging variants in genes required for microbicidal NADPH oxidase activity. Autosomal recessive p47phox-deficient CGD (p47-CGD) is predominantly caused by a two-nucleotide deletion in exon 2 (delGT) of NCF1. We developed PM359, an autologous CD34+ hematopoietic stem-cell therapy in which prime editing is used to correct delGT. Two participants received PM359 after myeloid conditioning with busulfan: neutrophils and platelets engrafted promptly in both patients. Adverse events were consistent with myeloid conditioning with busulfan. NADPH oxidase activity was observed in neutrophils within 1 month and was maintained for 6 months and 4 months as of the last follow-up visit in Participants 1 and 2, respectively. These results support further investigation of prime editing of CD34+ cells to treat p47-CGD. (Funded by Prime Medicine; ClinicalTrials.gov number, NCT06559176.).

Adolescent↗

PubMed2007

Genetics, biology and clinical management of myeloid cell primary immune deficiencies: chronic granulomatous disease and leukocyte adhesion deficiency.

PURPOSE OF REVIEW: Chronic granulomatous disease and leukocyte adhesion deficiency are the major primary immune deficiencies affecting phagocytic blood cells. Major advances in clinical diagnosis and development of novel treatments for these disorders merit review. RECENT FINDINGS: Clinically beneficial gene therapy correction of X-linked chronic granulomatous disease in two adult patients was reported. Nonmyeloablative busulfan conditioning before administration of gene corrected autologous hematopoietic stem cells was likely an essential maneuver to achieve successful gene therapy. There is an increased association of autoimmune disorders with chronic granulomatous disease. Preimplantation genetic diagnosis of leukocyte adhesion deficiency-I led to the birth of a normal child. A canine model of leukocyte adhesion deficiency-I facilitated development of new nonmyeloablative hematopoietic stem cell transplant and gene therapy approaches to leukocyte adhesion deficiency. Nonmyeloablative transplantation may provide an effective, but less toxic approach for leukocyte adhesion deficiency in children. There have been advances in understanding the basis of leukocyte adhesion deficiency-II and III. SUMMARY: The most important subjects reviewed in this chapter include new advances in development of gene therapy for chronic granulomatous disease and leukocyte adhesion deficiency-I; transplantation for leukocyte adhesion deficiency-I; prenatal diagnosis of leukocyte adhesion deficiency-I; and association of autoimmune diseases with chronic granulomatous disease.

Animals↗

PubMed2006

Analysis of human phagocyte flavocytochrome b(558) by mass spectrometry.

The catalytic core of the phagocyte NADPH oxidase is a heterodimeric integral membrane protein (flavocytochrome b (Cyt b)) that generates superoxide and initiates a cascade of reactive oxygen species critical for the host inflammatory response. In order to facilitate structural characterization, the present study reports the first direct analysis of human phagocyte Cyt b by matrix-assisted laser desorption/ionization and nanoelectrospray mass spectrometry. Mass analysis of in-gel tryptic digest samples provided 73% total sequence coverage of the gp91(phox) subunit, including three of the six proposed transmembrane domains. Similar analysis of the p22(phox) subunit provided 72% total sequence coverage, including assignment of the hydrophobic N-terminal region and residues that are polymorphic in the human population. To initiate mass analysis of Cyt b post-translational modifications, the isolated gp91(phox) subunit was subject to sequential in-gel digestion with Flavobacterium meningosepticum peptide N-glycosidase F and trypsin, with matrix-assisted laser desorption/ionization and liquid chromatography-mass spectrometry/mass spectrometry used to demonstrate that Asn-132, -149, and -240 are genuinely modified by N-linked glycans in human neutrophils. Since the PLB-985 cell line represents an important model system for analysis of the NADPH oxidase, methods were developed for the purification of Cyt b from PLB-985 membrane fractions in order to confirm the appropriate modification of N-linked glycosylation sites on the recombinant gp91(phox) subunit. This study reports extensive sequence coverage of the integral membrane protein Cyt b by mass spectrometry and provides analytical methods that will be useful for evaluating posttranslational modifications involved in the regulation of superoxide production.

Amino Acid Sequence↗

PubMed2006

WHIM syndrome myelokathexis reproduced in the NOD/SCID mouse xenotransplant model engrafted with healthy human stem cells transduced with C-terminus-truncated CXCR4.

WHIM(warts, hypogammaglobulinemia, recurrent bacterial infection, and myelokathexis) syndrome is a rare immunodeficiency caused in many cases by autosomal dominant C-terminal truncation mutations in the chemokine receptor CXCR4. A prominent and unexplained feature of WHIM is myelokathexis (hypercellularity with apoptosis of mature myeloid cells in bone marrow and neutropenia). We transduced healthy human CD34(+) peripheral blood-mobilized stem cells (PBSCs) with retrovirus vector encoding wild-type (wt) CXCR4 or WHIM-type mutated CXCR4 and studied these cells ex vivo in culture and after engraftment in a nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse xenograft model. Neither wt CXCR4 nor mutated CXCR4 transgene expression itself enhanced apoptosis of neutrophils arising in transduced PBSC cultures even with stimulation by a CXCR4 agonist, stromal cell-derived factor-1 (SDF-1 [CXCL12]). Excess wt CXCR4 expression by transduced human PBSCs enhanced marrow engraftment, but did not affect bone marrow (BM) apoptosis or the release of transduced leukocytes into PB. However, mutated CXCR4 transgene expression further enhanced BM engraftment, but was associated with a significant increase in apoptosis of transduced cells in BM and reduced release of transduced leukocytes into PB. We conclude that increased apoptosis of mature myeloid cells in WHIM is secondary to a failure of marrow release and progression to normal myeloid cell senescence, and not a direct effect of activation of mutated CXCR4.

Agammaglobulinemia↗

PubMed2006

Patients with chronic granulomatous disease have a reduced peripheral blood memory B cell compartment.

In this study, we have identified an altered B cell compartment in patients with chronic granulomatous disease (CGD), a disorder of phagocyte function, characterized by pyogenic infections and granuloma formation caused by defects in NADPH activity. This is characterized by an expansion of CD5-expressing B cells, and profound reduction in B cells expressing the memory B cell marker, CD27. Both findings were independent of the age, genotype, and clinical status of the patients, and were not accompanied by altered CD5 and CD27 expression on T cells. Focusing on CD27-positive B cells, considered to be memory cells based on somatically mutated Ig genes, we found that the reduction was not caused by CD27 shedding or abnormal retention of CD27 protein inside the cell. Rather, it was determined that CD27-negative B cells were, appropriately, CD27 mRNA negative, consistent with a naive phenotype, whereas CD27-positive B cells contained abundant CD27 mRNA and displayed somatic mutations, consistent with a memory B cell phenotype. Thus, it appears that CGD is associated with a significant reduction in the peripheral blood memory B cell compartment, but that the basic processes of somatic mutation and expression of CD27 are intact. X-linked carriers of CGD revealed a significant correlation between the percentage of CD27-positive B cells and the percentage of neutrophils with normal NADPH activity, reflective of the degree of X chromosome lyonization. These results suggest a role for NADPH in the process of memory B cell formation, inviting further exploration of secondary Ab responses in CGD patients.

Adolescent↗

PubMed2006

Polyclonal long-term MFGS-gp91phox marking in rhesus macaques after nonmyeloablative transplantation with transduced autologous peripheral blood progenitor cells.

We have recently reported that the RD114-pseudotyped MFGS-gp91phox vector achieves unprecedented levels of correction of the NADPH-oxidase gp91phox (approved gene symbol CYBB) defect in CD34(+) cells from patients with X-linked chronic granulomatous disease in the NOD/SCID mouse model. Considering clinical use of this vector, we transplanted autologous mobilized peripheral blood CD34(+) progenitor cells, transduced with the RD114-MFGS-gp91phox vector, into two healthy rhesus macaques following nonmyeloablative conditioning. The moderately high levels of in vivo marking seen in the first months following transduction decreased and stabilized at about 8 months posttransplant. Marking for both healthy animals after 15 months was 0.3 to 1.3 vector copies per 100 cells in lymphocytes, neutrophils, and monocytes. Vector insertion analyses performed by linear amplification-mediated PCR and sequencing identified 32 and 45 separate insertion sites in the animals. Identical insertion sites were found in myeloid cells and lymphocytes, demonstrating the successful transduction of lymphomyeloid progenitors. Some inserts landed in the vicinity of genes controlling cell cycle and proliferation. Statistical analyses of insertion sites 1 year posttransplant suggest a high diversity of insertion sites despite low marking.

Animals↗

PubMed2006

Sarcoidosis in chronic granulomatous disease.

In addition to increased susceptibility to infections in patients with chronic granulomatous disease (CGD), a higher incidence of sterile inflammatory disorders in these patients has been noted. However, sarcoidosis has not been reported previously in CGD. In this report, we describe two patients who have CGD and a disorder consistent with sarcoidosis on the basis of unequivocal clinical-radiographic presentations, their responses to treatment, and serum angiotensin-converting enzyme levels. Serum angiotensin-converting enzyme levels were measured in 26 other patients with CGD to establish an appropriate reference range. A possible relationship between CGD and sarcoidosis is discussed.

Adolescent↗

PubMed2006

Unique abnormalities of CD4(+) and CD8(+) central memory cells associated with chronic graft-versus-host disease improve after extracorporeal photopheresis.

Chronic graft-versus-host disease (cGVHD) remains a problematic complication of allogeneic hematopoietic stem cell transplantation. Laboratory parameters correlated with cGVHD have not been fully defined, although changes in CD4/CD8 ratios occur and a decrease in CD4(+) central memory T cells has been noted. Extracorporeal photopheresis (ECP) is an effective therapy for steroid-refractory cGVHD. We have noted changes in lymphocyte subsets after ECP. CD4(+) and CD8(+) T-cell central and effector memory populations were enumerated by flow cytometry in a cohort of 37 patients postallogeneic transplantation with symptomatic cGVHD. Of the patients with symptomatic cGVHD, 7 were treated with ECP over 6 months and prospectively assessed for changes in lymphocyte subsets. There was a highly significant correlation of an increase in CD8(+) central memory cells and a concomitant decrease in CD4(+) central memory cells in patients with symptomatic cGVHD. These changes were not detected in patients without cGVHD posttransplantation. In all, 7 patients with cGVHD followed up prospectively during ECP treatment showed a statistically significant normalization of the pattern of CD4(+) and a trend toward normalization of CD8(+) central memory T cells coincident with improvement of cGVHD. These data indicate a high correlation between disturbances in the balance of central and effector memory populations and cGVHD suggesting use in following up responses to therapy. The normalization of central and effector memory populations in response to ECP coincident with clinical improvement of cGVHD support a correlation between these laboratory parameters and cGVHD. Further studies are needed to demonstrate whether laboratory measurements of the magnitude of changes in central and effector memory populations are useful prognostically or can be used to guide response to therapy. The contrasting change in central memory cells (CD8(+) increased versus CD4(+) decreased) in cGVHD provide support for recent reports suggesting unique differences in the differentiation pathways for CD8(+) versus CD4(+) T cells.

Adult↗

PubMed2006

Gene therapy for immune disorders: good news tempered by bad news.

After a dozen years of human gene therapy trials characterized by minimal gene correction and disappointing clinical impact, the field of gene therapy received some good news in 2000. Infants with X-linked severe combined immunodeficiency who received retroviral gene addition to cells from their bone marrow developed impressive immune reconstitution. During the following 2 years, additional patients were treated and the news was even better-babies receiving gene therapy had sustained T-cell production and in several cases developed better cell function than most patients treated with standard bone marrow transplants. Unfortunately, bad news followed. Three of the patients experienced leukemic T-cell expansions, found to be associated with retroviral insertions into genomic DNA. Where does the field stand today?

Bone Marrow Transplantation↗

PubMed2005

Correction of canine X-linked severe combined immunodeficiency by in vivo retroviral gene therapy.

X-linked severe combined immunodeficiency (XSCID) is characterized by profound immunodeficiency and early mortality, the only potential cure being hematopoietic stem cell (HSC) transplantation or gene therapy. Current clinical gene therapy protocols targeting HSCs are based upon ex vivo gene transfer, potentially limited by the adequacy of HSC harvest, transduction efficiencies of repopulating HSCs, and the potential loss of their engraftment potential during ex vivo culture. We demonstrate an important proof of principle by showing achievement of durable immune reconstitution in XSCID dogs following intravenous injection of concentrated RD114-pseudotyped retrovirus vector encoding the corrective gene, the interleukin-2 receptor gamma chain (gamma c). In 3 of 4 dogs treated, normalization of numbers and function of T cells were observed. Two long-term-surviving animals (16 and 18 months) showed significant marking of B lymphocytes and myeloid cells, normalization of IgG levels, and protective humoral immune response to immunization. There were no adverse effects from in vivo gene therapy, and in one dog that reached sexual maturity, sparing of gonadal tissue from gene transfer was demonstrated. This is the first demonstration that in vivo gene therapy targeting HSCs can restore both cellular and humoral immunity in a large-animal model of a fatal immunodeficiency.

Animals↗

PubMed2005

Posaconazole as salvage therapy in patients with chronic granulomatous disease and invasive filamentous fungal infection.

Chronic granulomatous disease (CGD) is characterized by life-threatening bacterial and fungal infections. Treatment with posaconazole led to a complete response in 7 of 8 patients with CGD with invasive mold infections (7 proven cases and 1 possible case) after failure or intolerance of treatment with standard antifungal agents. In this preliminary study, salvage treatment with posaconazole was safe and effective.

Adolescent↗

PubMed2005

Improved transduction of human sheep repopulating cells by retrovirus vectors pseudotyped with feline leukemia virus type C or RD114 envelopes.

Gene therapy for hematopoietic diseases has been hampered by the low frequency of transduction of human hematopoietic stem cells (HSCs) with retroviral vectors pseudotyped with amphotropic envelopes. We hypothesized that transduction could be increased by the use of retroviral vectors pseudotyped with envelopes that recognize more abundant cellular receptors. The levels of mRNA encoding the receptors of the feline retroviruses, RD114 and feline leukemia virus type C (FeLV-C), were significantly higher than the level of gibbon ape leukemia virus (GaLV) receptor mRNA in cells enriched for human HSCs (Lin- CD34+ CD38-). We cotransduced human peripheral blood CD34+ cells with equivalent numbers of FeLV-C and GALV or RD114 and GALV-pseudotyped retroviruses for injection into fetal sheep. Analysis of DNA from peripheral blood and bone marrow from recipient sheep demonstrated that FeLV-C- or RD114-pseudotyped vectors were present at significantly higher levels than GALV-pseudotyped vectors. Analysis of individual myeloid colonies demonstrated that retrovirus vectors with FeLV-C and RD114 pseudotypes were present at 1.5 to 1.6 copies per cell and were preferentially integrated near known genes We conclude that the more efficient transduction of human HSCs with either FeLV-C- or RD114-pseudotyped retroviral particles may improve gene transfer in human clinical trials.

Amino Acid Transport System ASC↗

PubMed2005

Enhanced function with decreased internalization of carboxy-terminus truncated CXCR4 responsible for WHIM syndrome.

OBJECTIVE: WHIM (warts, hypogammaglobulinemia, recurrent bacterial infection, myelokathexis) syndrome is an autosomal dominant immune deficiency with severe chronic neutropenia and marrow neutrophil apoptosis. Carboxy-termini truncating mutations in the chemokine receptor CXCR4 have been identified in WHIM patients. We created a retrovirus encoding mutated CXCR4 (truncating point mutation 1000C-->T [R334X] inherited heterozygously in several WHIM patients) in order to transducer healthy human CD34 stem cells and K562 to overexpress mutated CXCR4 and determined its effect on receptor responses to stromal-derived factor-1 (SDF1). METHODS: Retrovirus vector was engineered to coexpress WHIM-associated R334X mutated CXCR4 together with green fluorescent protein (GFP). Control vectors included similar constructs with wild-type CXCR4 (WT-CXCR4) or only GFP. CD34+ cells and K562 were transduced with these vectors. Populations of 100% transduced K562 were established by sorting GFP+ cells by flow cytometry. We performed migration and calcium flux assays of transduced CD34+ cells and transduced/sorted K562. We also examined receptor recycling in response to SDF1. RESULTS: Healthy human CD34+ cells and/or human erythroleukemia K562 cells transduced to express mutated CXCR4, WT-CXCR4, or GFP alone demonstrated that mutated CXCR4 was associated with enhanced calcium flux and enhanced migration. There was also decreased receptor internalization and enhanced recovery of surface mutated CXCR4 in response to SDF1 compared with WT-CXCR4. CONCLUSION: We propose that decreased internalization of WHIM-associated mutated CXCR4 leads to prolongation/enhancement of signaling in response to SDF1 and that this may provide the biochemical basis for the autosomal dominant abnormalities of cell trafficking and function associated with WHIM syndrome.

Agammaglobulinemia↗

PubMed2005

HIF-1alpha: a master regulator of innate host defenses?

In the days following infection, when the human body develops and refines antibodies and prepares to mount an adaptive immune response, the bulwark of innate host defense against microbial infection is the polymorphonuclear leukocyte (PMN). PMNs seek out, identify, engulf, and sterilize invading microbes using both O2-dependent and O2-independent antimicrobial systems. A decrease in PMN numbers or function caused by immunosuppression or disease increases the risk of infection. In this issue of the JCI, Peyssonnaux et al. identify a novel and essential role for hypoxia-inducible factor-1alpha in regulating several important PMN functions relevant to host defense, including transcription of cationic antimicrobial polypeptides and induction of NO synthase.

Animals↗

PubMed2004

Cloning and functional analysis of the rhesus macaque ABCG2 gene. Forced expression confers an SP phenotype among hematopoietic stem cell progeny in vivo.

Hematopoietic cells can be highly enriched for repopulating ability based upon the efflux of the fluorescent Hoechst 33342 dye by sorting for SP (side population) cells, a phenotype attributed to expression of ABCG2, a member of the ABC transporter superfamily. Intriguingly, murine studies suggest that forced ABCG2 expression prevents hematopoietic differentiation. We cloned the full-length rhesus ABCG2 and introduced it into a retroviral vector. ABCG2-transduced human peripheral blood progenitor cells (PBPCs) acquired the SP phenotype but showed significantly reduced growth compared with control. Two rhesus macaques received autologous PBPCs split for transduction with the ABCG2 or control vectors. Marking levels were similar between fractions with no discrepancy between bone marrow and peripheral blood marking. Analysis for the SP phenotype among bone marrow and mature blood populations confirmed ABCG2 expression at levels predicted by vector copy number long term, demonstrating no block to differentiation in the large animal. In vitro studies showed selective protection against mitoxantrone among ABCG2-transduced rhesus PBPCs. Our results confirm the existence of rhesus ABCG2, establish its importance in conferring the SP phenotype, suggest no detrimental effect of its overexpression upon differentiation in vivo, and imply a potential role for its overexpression as an in vivo selection strategy for gene therapy applications.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed2004

Long-term interferon-gamma therapy for patients with chronic granulomatous disease.

BACKGROUND: Chronic granulomatous disease (CGD) is a rare disorder of phagocytes in which absent production of superoxide and hydrogen peroxide in phagocytes predisposes patients to bacterial and fungal infections. Infections are dramatically reduced by prophylaxis with antibiotics, antifungals, and interferon- gamma (IFN-gamma ). METHODS: Seventy-six patients with CGD were enrolled in an uncontrolled, open-label follow-up study to assess the long-term clinical safety and efficacy of IFN-gamma therapy. Patients received IFN-gamma subcutaneously 3 times per week. RESULTS: We observed patients for up to 9 years, for a total observation period of 328.4 patient-years. The incidence of serious infections was 0.30 infections per patient-year; for serious bacterial infections, the incidence was 0.18 cases per patient-year, and for serious fungal infections, it was 0.12 cases per patient-year. Thirty-seven percent of patients reported an adverse event, the most common of which was fever. Twenty-six patients withdrew from the study (3 because of adverse events, 15 because of patient preference, and 8 because of transfer to another trial). There were no life-threatening IFN-gamma-related adverse events and no discernible effects on growth. The overall mortality rate was 1.5% per patient-year. CONCLUSION: IFN-gamma prophylaxis for CGD appears to be effective and well tolerated over a prolonged period of time.

Adolescent↗

PubMed2004

Severe chronic graft-versus-host disease is characterized by a preponderance of CD4(+) effector memory cells relative to central memory cells.

Donor alloreactive CD4(+) T cells are important to the pathogenesis of chronic graft-versus-host disease (cGVHD), but specific subsets of CD4(+) T cells responsible for GVHD have not been defined. We hypothesized that cGVHD might be associated with a preponderance of CD4(+) effector memory cells (CCR7(-)/CD62L(low), CD4(EM)). We analyzed CCR7 and CD62L expression on CD4(+) T cells from stem cell transplantation patients, who did or did not develop cGVHD, and healthy donors. Patients with cGVHD had a higher percentage of CD4(EM) cells (35.5% +/- 2.9%) than healthy donors (13.8% +/- 0.7%; P <.0001) or patients without cGVHD that received a transplant (21.7% +/- 2.1%; P <.01). Using corticosteroid dose as a surrogate marker for cGVHD severity, severe cGVHD was associated with a higher percentage of CD4(EM) cells. The proportion of CD4(EM) cells in corticosteroid-dependent patients with systemic lupus erythematosis or Wegener granulomatosis did not differ from patients without cGVHD that received a transplant. This finding implies that overrepresentation of CD4(EM) cells is a unique feature of cGVHD.

CD4-Positive T-Lymphocytes↗

PubMed2004

Gene expression profiling provides insight into the pathophysiology of chronic granulomatous disease.

Human polymorphonuclear leukocytes (PMNs or neutrophils) kill invading microorganisms with reactive oxygen species (ROS) and cytotoxic granule components. PMNs from individuals with X-linked chronic granulomatous disease (XCGD) do not produce ROS, thereby rendering these individuals more susceptible to infection. In addition, XCGD patients develop tissue granulomas that obstruct vital organs, the mechanism(s) for which are unknown. To gain insight into the molecular processes that contribute to the pathophysiology of XCGD, including formation of granulomas, we compared global gene expression in PMNs from XCGD patients and healthy control individuals. Genes encoding mediators of inflammation and host defense, including CD11c, CD14, CD54, FcgammaR1, FcalphaR, CD120b, TLR5, IL-4R, CCR1, p47(phox), p40(phox), IL-8, CXCL1, Nramp1, and calgranulins A and B, were up-regulated constitutively in unstimulated XCGD patient PMNs. By comparing transcript levels in normal and XCGD PMNs after phagocytosis, we discovered 206 genes whose expression changed in the presence and the absence of ROS, respectively. Notably, altered Bcl2-associated X protein synthesis accompanied defective neutrophil apoptosis in XCGD patients. We hypothesize that granuloma formation in XCGD patients reflects both increased proinflammatory activity and defective PMN apoptosis, and we conclude that ROS contribute directly or indirectly to the resolution of the inflammatory response by influencing PMN gene transcription.

Adolescent↗