Base Editing for Mutation Repair in Hematopoietic Stem & Progenitor Cells for X-Linked Chronic Granulomatous Disease

Study Description:

Open-label, phase 1/2 trial to determine the safety, and efficacy of a single infusion of base-edited (BE) autologous hematopoietic stem and progenitor cells (HSPCs) for treatment of X-linked chronic granulomatous disease (X-CGD). Base editing is performed to repair CYBB missense gene mutations (eg, CYBB c.676C>T). The study hypotheses are that 1) base editing can efficiently repair gene mutations in HSPCs; and 2) BE HSPCs can engraft and differentiate into functional phagocytes with restored nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity.

During the initial development phase each study participant will undergo apheresis for CD34+ HSPC collection for the development and validation of a mutation-specific BE system.

During the treatment phase, participants will receive a one-time infusion of the BE autologous HSPC (study product) after the administration of busulfan conditioning (total 12 mg/kg with a targeted total AUC of 65,000 ng/mL x hr). Sirolimus will be initiated at Day -1 and will be given for approximately 3-6 months.

Participants will have follow-up evaluations at months 3, 6, 12, 18, and 24, and yearly thereafter until 5 years after treatment. Key study assessments include adverse event (AE) assessment, blood laboratory evaluations of functional protein made from the target gene (CYBB, encodes for gp91^phox), and deoxyribonucleic acid (DNA) sequencing to identify rates of gene repair and off-target mutation.

The final study follow-up under this protocol will be at 5 years, but long-term follow-up under a separate NIH protocol will continue annually to 15 years after treatment.

Objectives:

Primary Objectives:

• To evaluate the safety of BE autologous CD34+ cells.
• To evaluate the efficacy of BE autologous CD34+ cells.

Secondary Objectives:

-To evaluate:

• Efficiency of base editing (percentages of target nucleotide conversion).
• Engraftment capability of BE HSPCs.
• Efficiency in restoring gp91^phox expression
• Efficiency in restoring NADPH oxidase function.
• Clinical efficacy.
• Stability of gene correction.

Exploratory Objectives:

-To evaluate:

• Specificity of base editing by assessing rates of off-target edits at top predicted sites.
• Random off-target edits by whole genome sequencing (WGS).

Endpoints:

Primary Endpoints:

• Safety of gene therapy using BE autologous HSPCs as measured by study product-related AEs and serious AEs (SAEs) through 2 years after treatment.
• Efficacy of gene therapy determined as percentages of subjects who have >= 10% oxidase-positive granulocytes at 12 months after infusion.

Secondary Endpoints:

• Determine frequency of converted target nucleotide alleles.
• Assess the frequency of gene-modified alleles compared to infusion product in peripheral blood at 12 months.
• Evaluate efficacy of restoring gp91^phox expression.
• Evaluate efficacy of NADPH oxidase functional restoration as measured by the dihydrorhodamine (DHR) flow cytometry oxidase assay at 12 months after treatment (>=10% oxidase-positive neutrophils), or by quantitative measurement of reactive oxygen species (ROS) using the ferricytochrome C assay on peripheral blood granulocytes.
• Evaluate clinical benefit by improvement of baseline clinical problems such as recurrent infections, growth failure, malnutrition, inflammatory bowel disease, or antibiotic usage.
• Assess stability of gene correction by serial measurement of percent of nucleotide-converted alleles.

Exploratory Endpoints:

• Evaluate specificity by assessing for off-target edits at predicted sites with high-throughput sequencing of the 5 most common candidate off-target loci identified by a comprehensive in vitro screen, Circularization for High-throughput Analysis of Nuclease Genome-wide Effects by sequencing (CHANGE-seq-BE), in peripheral blood cells.
• Determine rate of random off-target edits WGS.

• INCLUSION CRITERIA:

->= 18 years of age.

• Confirmed CYBB c.676 C>T mutation.
• Male patients.
• Clinically stable and eligible to undergo apheresis and conditioning chemotherapy.

->=5 x 10^6 cryopreserved cells/kg body weight available for study product manufacturing.

• History of at least one prior serious infection or inflammatory complication requiring hospitalization despite conventional therapy.
• In the experience of a qualified clinical investigator, the patient has a poor prognosis.
• Able and willing to use a highly effective method of contraception, AND partner has communicated her willingness through subject to do same, if engaging in potentially reproductive sex from the signing of the informed consent and for 6 months after IMP infusion. Acceptable methods of contraception include the following:

• Hormonal contraception in continuously effective use by female partner.
• Male or female condom with spermicide as indicated.
• Diaphragm or cervical cap in consistent and effective pattern of use with a spermicide by female partner.
• Intrauterine device in-situ throughout above period by female partner.

Exclusion criteria

Individuals meeting any of the following criteria will be excluded from study participation:

• Untreated, acute infection.
• Elevated anti-gp91 specific autoantibodies >2 x ULN
• Elevated anti-gp91 specific T cells (>10 fold)
• Anti-platelet antibody screening with >1 anti-platelet antibody positive in the presence of an ongoing brain infection; OR >1 anti-platelet antibody positive and considered unsafe for study participation after consultation with hematology specialist.
• Known hypersensitivity to busulfan or any component of the product.
• Contraindications for administration of busulfan.
• Any current or pre-existing hematologic malignancy.
• Chronic infections that are considered unsafe for participation in the study by Infectious Disease Consultant.
• Cardiac abnormalities and neurological abnormalities that are deemed unsafe to participate in the study.
• Childhood malignancy (occurring before 18 years of age) in the patient or a first degree relative, or previously diagnosed known genotype of the participant conferring a predisposition to cancer (no DNA or other testing for cancer predisposition genes will be performed as part of the screen for this protocol).
• Hematological parameters unsafe for apheresis or above Grade 2 Common Terminology Criteria for Adverse Events (CTCAE) criteria until improved.
• Hepatic dysfunction- alanine aminotransferase (ALT >3.0 - 5.0 x upper limit of normal [ULN]), aspartate aminotransferase (AST >3.0 - 5.0 x ULN), bilirubin (>1.5 - 3.0 x ULN).
• Renal dysfunction-serum creatinine >1.5 - 3.0 x ULN or creatinine clearance 59-30 mL/min/1.73 m^2.
• Coagulation dysfunction- Prothrombin INR or Partial thromboplastin time >2 x ULN (patients on controlled anticoagulation agents will not be excluded for therapeutic levels).
• Uncontrolled hypertension- Systolic BP 140-159 mm Hg or diastolic BP 90-99 mm Hg.
• Abnormal blood chemistries- Hyperkalemia (K >5.5 - 6.0 mmol/L), Hypokalemia (<LLN - 3.0 mmol/L and requiring intervention); OR Hypercalcemia (corrected serum calcium >11.5 - 12.5 mg/dL), Hypocalcemia (corrected serum calcium <8.0 -7.0 mg/dL)

These values exclude false abnormalities secondary to hemolysis.

• Cytogenetic abnormalities evidenced on bone marrow aspirate.
• Pulmonary dysfunction FEV1<25% predicted.
• Previous treatment with gene therapy or gene editing products.
• Previous receipt of non-HLA matched donor granulocyte transfusions.
• Any other condition that, in the opinion of the investigator, may unduly compromise the safety or compliance of the patient, or would make successful study completion highly unlikely.