Ex Vivo Lentiviral Hematopoietic Stem Cell (HSC) Gene Therapy May Represent a Curative Therapy for the Life-Threatening Inborn Error of Immunity Severe Combined Immunodeficiency Due to Adenosine Deaminase (ADA) Deficiency (ADA-SCID)

Speaker: Katelyn Masiuk, University of California, Los Angeles

Key Highlights       

ADA-SCID is a rare genetic disorder caused by the absence of the ADA enzyme, which is crucial for immune function. The gene therapy involves harvesting the patient's autologous HSCs, which are genetically modified using a third-generation lentiviral vector to deliver the ADA gene. After conditioning with low-dose busulfan, the modified cells are reinfused into the patient.

Study Design and Patient Cohorts: Data Source: Pooled data from four Phase 1/2 trials (2012-2019) conducted at UCLA and University College London.

Cohort Details:

• 62 patients in total, with a median age at treatment of 10 months (U.S.) and 14 months (U.K.).
• Cell sources: Bone marrow (U.S.) and mobilized peripheral blood (U.K.).
• 38 patients received fresh HSC products, 21 received cryopreserved, and 3 received a combination.

Efficacy Results:

1. Overall survival (OS) and Event-Free Survival (EFS):
   1. 100% OS: 11 years (U.K.) and 10 years (U.S.).
   2. 95% EFS (events: reinstitution of ERT or need for allogeneic transplant).
   3. Three cases of early treatment failure due to insufficient gene marking were managed successfully with alternative treatments.
2. Immune Reconstitution:
   1. Robust and sustained T-cell reconstitution to near-normal levels within 2 years post-gene therapy.
   2. B-cell function restored, with 98% of patients discontinuing IgG replacement within 12 months.
3. Vaccine response: Protective antibody titres achieved in all evaluated patients following tetanus vaccination.
4. Biomarkers:
   1. Stable ADA enzyme production and reduction of toxic metabolites to safe levels.
   2. Durable gene marking in granulocytes and peripheral blood mononuclear cells.

Safety Profile: Excellent long-term safety with no clonal expansion or malignancies observed.

Conclusion:

Ex vivo lentiviral HSC gene therapy offers a curative treatment for ADA-SCID, providing robust, durable immune reconstitution and an excellent safety profile. With regulatory approval anticipated, this therapy has the potential to transform the treatment landscape for ADA-SCID.

Factor VIII Expression from a Novel F8 Transgene through a Lentiviral Vector Transduced CD34+ Autologous Hematopoietic Stem Cells for Gene Therapy of Severe Hemophilia Α: Final Results from a Phase 1 Clinical Trial

Speaker: Alok Srivastava, Christian Medical College, India

Key Highlights

Current gene therapy for hemophilia primarily uses adeno-associated viral (AAV) vectors, which have led to the approval of several products, including one for hemophilia A. However, it has notable limitations: the expression levels can be unpredictable, and they tend to drop after 6-12 months, particularly in hemophilia A, often accompanied by trans-aminitis requiring immunosuppression. These challenges highlight the need for alternative gene therapy approaches for hemophilia.

Study Design and Methods:

• Gene Therapy Candidate: A 3rd-generation, self-inactivating lentiviral vector delivering a bioengineered FVIII transgene (8083) driven by a CD68 promoter, enabling sustained FVIII production in differentiated blood cells.
• Study Population: Included young adults with severe hemophilia A, high annualized bleeding rates, and no FVIII inhibitors, with prior exposure to FVIII therapy (>100 infusions).

Treatment Process:

• Stem Cell Collection: Mobilization using G-CSF and Plerixafor to obtain sufficient CD34+ cells.
• Ex Vivo Transduction: Using a lentiviral vector, with modifications after initial patients to include a transduction enhancer.
• Transplantation: Reduced-toxicity myeloablative conditioning with treosulfan and fludarabine, followed by infusion of transduced HSCs.

Efficacy Results:

1. Factor VIII Activity:
   1. Median FVIII activity stabilized post-treatment at ~5.1% (Group 1) and 20-40% (Group 2), with peak activity >50% in Group 2 participants.
   2. FVIII levels remained stable beyond 4-6 months of follow-up.
2. Bleeding Events:
   1. No spontaneous bleeding occurred post-therapy, irrespective of FVIII activity levels.
   2. Two participants reported no bleeding even after trauma, including one with a head injury.
3. Clinical Outcomes: Sustained hemostasis, with long-term expression attributed to genome integration of the vector.

Safety Profile:

1. Conditioning Toxicity: Mucositis was mild (≤Grade 2), and patients maintained oral intake during the peri-transplant period.
2. Fertility: Normal sperm counts were observed post-treatment in all tested participants, with only one reporting low motility.

Key Insights and Innovations:

1. Advantages Over AAV Therapy:
   1. Addresses limitations such as transaminitis, pre-existing AAV antibodies, and restricted eligibility for pediatric patients due to liver maturity.
   2. Provides sustained FVIII expression due to the vector's genome integration capability.
2. Group-Specific Differences: Enhanced FVIII expression in Group 2 participants resulted from protocol improvements, particularly the addition of a transduction enhancer.
3. Global Implications: The low-cost autologous transplantation process (<$10,000 in India) highlights the potential for this approach to make gene therapy more accessible globally.

Conclusion:

This Phase 1 trial established the safety and feasibility of lentiviral vector-mediated gene therapy for hemophilia A, with sustained FVIII expression and excellent clinical outcomes. While the procedure remains complex, the results are promising for broader implementation in regions with constrained resources. Further trials will refine the approach and evaluate long-term outcomes.

Efficacy and Safety of Giroctocogene Fitelparvovec in Adults with Moderately Severe to Severe Hemophilia Α: Primary Analysis Results from the Phase 3 ΑFFINE Gene Therapy Trial

Speaker: Andrew Leavitt, UCSF Adult Hemophilia Treatment Centre

Key Highlights       

The Phase 3 AFFINE trial evaluated the efficacy and safety of giroctocogene fitelparvovec, a gene therapy using an AAV6 vector to deliver a B-domain-deleted human FVIII cDNA for adults with moderately severe to severe hemophilia A (FVIII levels ≤1%).

The trial involved:

• A 6-month lead-in period to collect baseline data on annualized bleeding rates (ABR) and FVIII usage.
• A single infusion of the gene therapy followed by monitoring for 5 years.
• Inclusion criteria required participants to have no AAV6 antibodies, no FVIII inhibitors, and acceptable liver function.
• The primary endpoint of the trial was the total annualized bleeding rate (ABR), assessed after at least 15 months of follow-up.
• Key secondary endpoints included treated ABR and the use of factor replacement therapy post-infusion.
• Of the 241 patients screened, 75 received the therapy, with 50 included in the efficacy analysis (≥15 months follow-up).

Efficacy Outcomes:

1. Reduction in Bleeding Events:
   1. Total ABR decreased significantly from 4.65 during the lead-in period to 0.26 post-therapy (excluding one outlier).
   2. 64% of participants reported no bleeding events.
   3. Treated ABR decreased from 4.08 to 0.07, with 88% of participants reporting no treated bleeds.
2. FVIII Activity:
   1. FVIII levels exceeded 5% for all participants by 12 weeks and remained above 50% on average at 1 and 2 years.
   2. FVIII activity was stable after an initial rise, showing minimal variability throughout the two-year follow-up period.
3. Factor Use: The annualized infusion rate for prophylactic FVIII replacement therapy dropped significantly from 124 infusions to 0.21.

Safety Profile:

1. Adverse Events (AEs):
   1. 74 out of 75 participants experienced AEs, with 15 classified as serious AEs.
   2. No cases of FVIII inhibitors or therapy-related malignancies were reported.
2. Thrombotic Events:
   1. One patient with a prior history of thrombosis experienced a thrombotic event.
   2. Elevated FVIII levels (>150%) were observed in 37 participants; 30% were managed with prophylactic anticoagulants, which were effective in preventing thrombotic complications.
3. Liver Enzyme Elevations: ALT elevations occurred in 47 participants, managed with corticosteroids, which were well-tolerated. By the study's cutoff, no participants remained on corticosteroids.

Conclusion:

Giroctocogene fitelparvovec showed significant efficacy in reducing ABR and FVIII usage, with sustained FVIII expression. The safety profile, including thrombotic risk and liver enzyme elevation, was manageable. These findings support the potential of gene therapy to revolutionize the treatment of hemophilia A.

Durability and Safety of Factor VIII Expression Post AAV in Adult Hemophilia A Mice

Speaker: Lindsey George, University of Pennsylvania

Key Highlights       

This session highlighted a preclinical study on the long-term effects of AAV-mediated Factor VIII (FVIII) expression in adult hemophilia A mice. The study aimed to evaluate the durability of FVIII expression, liver health, and potential tumorigenesis risks following AAV gene therapy.

Study Design:

• Objective: Assess long-term FVIII expression durability, liver health, and tumorigenesis risks.
• Model: Adult hemophilia A mice treated with AAV8 vectors expressing mild, normal, and elevated FVIII levels.
• Follow-up: Mice were monitored for up to 72 weeks with regular assessments of FVIII levels, liver histology, and markers of hepatocyte stress.

Key Findings:

1. Durability of FVIII Expression: Mild and Normal Expression: FVIII levels remained stable over 72 weeks.
2. Elevated Expression: Significant decline in FVIII levels between 8 and 72 weeks, associated with reductions in liver vector copy number. No gene silencing observed.
3. Hepatic Stress and Vector Copy Number:
   1. Elevated FVIII expression induced endoplasmic reticulum (ER) stress, evidenced by increased CHOP mRNA expression at weeks 8 and 72.
   2. ER stress likely contributed to hepatocyte turnover and vector copy loss, accompanied by transient ALT elevations corresponding to FVIII expression declines.
4. Mechanistic Insights:
   1. Loss of FVIII expression was linked to the unfolded protein response, resulting from inefficient FVIII secretion in hepatocytes.
   2. Moderate FVIII expression appears to minimize ER stress and hepatocyte turnover, making it a more durable therapeutic target.

Implications for AAV Gene Therapy:

Enhanced Durability: Moderate FVIII expression (similar to mild hemophilia A) ensures durable therapeutic effects without inducing detrimental ER stress. Enhanced-function FVIII variants could further reduce the required protein amount, minimizing hepatocyte stress.

Long-Term Liver Safety: No liver toxicity or tumorigenesis observed, providing confidence in the long-term safety of AAV-mediated FVIII gene therapy. However, ongoing monitoring is necessary due to the dynamics of FVIII secretion and hepatocyte turnover.

Conclusion:

This study demonstrated the feasibility of long-term AAV-mediated FVIII expression in hemophilia A mice, with no liver toxicity or tumorigenesis. Moderate FVIII expression ensures durable therapeutic efficacy while minimizing ER stress. These findings pave the way for clinical testing of optimized FVIII variants in AAV-based gene therapies.

Site-Specific Insertion of Factor VIII Gene Results in Durable Factor VIII Expression in Nonhuman Primates

Speaker: Alan Brooks, Head of Preclinical, Metagenomi

Key Highlights

The session was about a study on using CRISPR-mediated gene editing to insert the Factor VIII (FVIII) gene into nonhuman primates (NHPs), offering a potential durable and curative treatment for hemophilia A.

Study Objective: To develop a gene-editing-based therapy to achieve durable FVIII expression and overcome challenges faced by AAV therapies.

Methodology:

• Conducted in cynomolgus macaques with a modified FVIII gene to minimize immune responses.
• FVIII levels measured up to 16 months, with liver biopsies taken to assess integration frequencies.
• CRISPR Type 5 nuclease creates a double-strand break in the albumin gene’s intron 1, allowing AAV to deliver the FVIII gene, while lipid nanoparticles (LNPs) provide the CRISPR machinery for editing, with FVIII expression driven by the albumin promoter for sustained, high expression.

Key Findings:

1. Therapeutic Durability of FVIII Expression:
   1. All NHPs achieved therapeutic FVIII levels (8%, 30%, and 75% of normal activity).
   2. FVIII expression remained stable over 16 months, with no evidence of silencing.
   3. Integration frequency positively correlated with FVIII expression levels.
2. Safety:
   1. Liver Function: Transient ALT elevations were observed post-infusion but returned to baseline within a week. No disruptions to albumin gene function were noted.
   2. Health Status: Animals showed normal weight gain and remained healthy throughout the study.
   3. Ongoing studies will further assess liver histology and long-term tumorigenesis risks.
3. Off-Target Analysis: No off-target edits detected after screening 481 potential sites, with nearly 100% on-target editing efficiency.
4. Bioengineered FVIII Variant:
   1. An engineered FVIII variant demonstrated enhanced activity and higher plasma levels compared to wild-type FVIII.
   2. This variant is expected to reduce AAV dose requirements, lowering immunogenicity and corticosteroid use in clinical settings.

Conclusion:

Dr. Brooks’s study highlights the potential of CRISPR-based gene editing for hemophilia A, showing durable and precise FVIII expression through integration at the albumin locus. The use of a bioengineered FVIII variant could further enhance safety and reduce the therapeutic dose in future clinical applications.

Using Gene Therapy to Solve Challenges with CAR-T Cell Immunotherapy: Lead Selection and Preclinical Development of an Adeno-Associated Virus with Reduced Immunogenicity Exhibiting Efficient and Long-Term Expression of an Anti-CD19 T-Cell Engager

Speaker: Timothy Cripe, Nationwide Children’s Hospital

Key Highlights

Key Challenges in CAR-T Therapy:

CAR-T therapy, although effective, has several key limitations, including complex and individualized manufacturing, toxicity risks such as cytokine release syndrome (CRS) and neurotoxicity, and short-term persistence of CAR-T cells. Moreover, CAR-T's effectiveness depends on high target density and selective T-cell subsets. VNX-101 seeks to address these challenges by leveraging the enduring expression of an anti-CD19 T-cell engager through AAV, mimicking CAR-T therapy while mitigating associated risks.

Development of VNX-101:

VNX-101 uses a bispecific T-cell engager, similar to blinatumomab, which bridges T-cells and tumor cells to facilitate immune synapse formation. Key advantages over CAR-T therapy include:

• Off-the-shelf availability: No need for individualized manufacturing.
• No conditioning chemotherapy: Simplifies the treatment process.
• Reduced CRS incidence: Gradual onset of activity leads to a lower risk of severe CRS.
• Broader T-cell engagement: Increases efficacy across a wider population.

Preclinical Development: Preclinical studies demonstrated the promising efficacy of VNX-101:

• Efficacy: In humanized mouse models, VNX-101 successfully eliminated leukemia cells and suppressed tumor progression. The therapy also provided lasting protection, as shown in a delayed challenge model.
• Safety: Toxicology studies in mice and baboons showed no significant adverse effects. In baboons, transient liver enzyme elevations were noted but resolved spontaneously, indicating a favorable safety profile.

First-in-Human Clinical Trial (SENTRY-ALL):

VNX-101 is now being tested in a Phase 1/2 clinical trial (SENTRY-ALL) to evaluate its safety and efficacy in patients with relapsed/refractory CD19-positive ALL. The trial focuses on patients with low disease burden (<5% bone marrow blasts), with a dose-escalation study to determine optimal doses (e11–e12 vector genomes/kg).

Conclusion:

VNX-101 represents a significant advancement in immunotherapy, combining the long-lasting therapeutic effects of gene therapy with the immune engagement seen in CAR-T cells. This approach holds the potential for transforming the treatment landscape for CD19-positive ALL and other B-cell-mediated diseases. The ongoing clinical trial is critical to confirming the preclinical findings and determining its broader clinical applicability.

ASH Annual Meeting and Exposition, 7-10 December 2024, San Diego, California