Study of Gene Therapy Using a Lentiviral Vector to Treat X-linked Chronic Granulomatous Disease

The therapeutic product to be evaluated is autologous CD34+ hematopoietic stem cells (HSC) modified by ex vivo transduction using the pCCLchimGP91WPRE lentiviral vector (G1XCGD Modified Autologous BM CD34 cells) containing the human CGD gene. The G1XCGD lentiviral vector is a 3rd generation self-inactivating lentiviral vector which directs gp91phox expression from a codon-optimized form of the CYBB gene preferentially to myeloid cells, with a modified WPRE (PRE4).

G1XCGD is an integrative, 3rd generation replication-defective, self-inactivating (SIN) HIV-derived Lentiviral (LV) vector, with a mutated Woodchuck hepatitis virus Posttranscriptional Regulatory Element (WPRE) sequence. A LV vector derived from HIV-1 has been chosen with respect to LV natural properties: they are genetically stable, permanently integrate into the genome of transduced cells and provide long-term gene expression in vitro and in vivo. The transduction of Hematopoietic Stem Cells (HSC) with such LV can be achieved after limited pre-activation of the cells in short-term cultures with cytokines, in conditions that are compatible with the preservation of the self-renewing capacities of these cells. These properties make these LV suitable for ex-vivo gene therapy strategies using HSC.

G1XCGD provirus includes a chimeric promoter designed to regulate the transgene expression in myeloid cells and a transgene called GP91 (also known as CYBB), which is a codon-optimized cDNA sequence of the human CYBB gene also known as GP91-PHOX or NOX2 gene. The promoter is a synthetic chimeric element created by the fusion of c-Fes and Cathepsin G minimal 5'-flanking regions. Cathepsin G is a serine protease stored in the azurophil granules of neutrophil granulocytes. Part of the chimeric promoter contains binding sites for myeloid transcription factors C/EBP and PU.1 from the upstream region of the transcription start site of the Cathepsin G gene. The other part of the chimeric promoter is a human c-Fes sequence that has been added to enhance the Cathepsin G promoter activity in granulocytic cells. The resulting chimeric promoter is able to i) regulate the expression of the GP91 transgene in myeloid cells in a specific manner, and ii) to effectively restore NADPH-oxidase activity in granulocytes, as reported by Santilli et al. (Santilli et al., 2011) and confirmed in preclinical studies conducted with the G1XCGD vector. The GP91 transgene codes for the 570 amino-acid cytochrome b-245, a 91 kD beta polypeptide that is also known as the NADPH-oxidase catalytic subunit gp91-phox, or cytochrome b-245 heavy chain, or gp91-phox protein.

Inclusion criteria

(Part A & B)

• Male X-CGD patients > 23 months of age
• Molecular diagnosis confirmed by DNA sequencing and supported by laboratory evidence for absent or reduction > 95% of the biochemical activity of the NADPH-oxidase
• At least one prior, ongoing or refractory severe infection and/or inflammatory complications requiring hospitalization despite conventional therapy
• No 10/10 HLA-matched donor available after initial search of NMDP registries
• No co-infection with Human Immunodeficiency Virus (HIV)-1 or -2, hepatitis B virus or hepatitis C virus, adenovirus, parvovirus B 19 or toxoplasmosis, or active infection with CMV
• Written informed consent for adult patient, and assent for pediatric subjects seven years or older.
• Parental/guardian and, where appropriate, child's signed consent/assent

Exclusion criteria

• Age < 23 months
• 10/10 HLA identical (A,B,C,DR,DQ) family or unrelated or cord blood donor unless there is deemed to be an unacceptable risk associated with an allogeneic procedure
• Contraindication for leukapheresis or bone marrow harvest (anemia Hb <8g/dl, cardiovascular instability, severe coagulopathy)
• Appropriate organ function as outlined below must be observed within 8 weeks of entering this trial.

• Hematologic

• Anemia (hemoglobin < 8 g/dL).
• Neutropenia (absolute granulocyte count <1,000/mm3)
• Thrombocytopenia (platelet count < 150,000/mm3).
• PT or PTT > 2X the upper limits of normal (patients with a correctable deficiency controlled on medication will not be excluded).
• Cytogenetic abnormalities known to be associated with hematopoietic defect on peripheral blood or bone marrow.
• Infectious

a. Evidence of co-infection with HIV-1, HIV-2, hepatitis B, Hepatitis C, adenovirus, parvovirus B19, toxoplasmosis. CMV infection is allowable as long as the infection is under control.

• Pulmonary

a. Resting O2 saturation by pulse oximetry < 90% on room air.

• Cardiac

• Abnormal electrocardiogram (EKG) indicating cardiac pathology.
• Uncorrected congenital cardiac malformation with clinical symptomatology.
• Active cardiac disease, including clinical evidence of congestive heart failure, cyanosis, hypotension.
• Poor cardiac function as evidenced by LV ejection fraction < 40% on echocardiogram.
• Neurologic

• Significant neurologic abnormality by examination.
• Uncontrolled seizure disorder.
• Renal

• Renal insufficiency: serum creatinine ≥ 1.5 mg/dl, or ≥ 3+ proteinuria.
• Abnormal serum sodium, potassium, calcium, magnesium, phosphate at grade III or IV by the Common Terminology Criteria for Adverse Events (CTCAE) version 4.0.
• Hepatic/GI:

• Serum transaminases > 5X the upper limit of normal (ULN).
• Serum bilirubin > 2X ULN.
• Serum glucose > 1.5x ULN.
• Oncologic

a. Evidence of active malignant disease

• General

• Expected survival < 6 months
• Major congenital anomaly
• Ineligible for autologous HSCT by the criteria at the clinical site.
• Contraindication for administration of conditioning medication. (Known sensitivity to Busulfan)
• Administration of gamma-interferon within 30 days before the infusion of transduced, autologous CD34+ cells.
• Participation in another experimental therapeutic protocol within 6 months prior to baseline and during the study period.
• Tested positive (definitive) for the presence of multiple types (2 or more) of anti-platelet antibodies.
• Any other condition that, in the opinion of the Investigator, may compromise the safety or compliance of the patient or would preclude the patient from successful study completion.
• Patient/Parent/Guardian unable or unwilling to comply with the protocol requirements.

Part B Additional exclusion criteria:

• Patients >12 years of age at enrolment
• Patients ≤12 years of age with a body weight >40kg at enrolment