Hematopoietic reconstitution after myeloablative conditioning and transplantation may be entirely driven by hematopoietic stem cells (HSCs), according to a preclinical study presented at the 

2022 American Society of Gene and Cell Therapy Annual Meeting

, taking place May 16-19, 2022, in Washington, DC.

“These findings have major implications for HSC transplantation and particularly for HSC gene therapy,” reported Stefan Radtke, PhD, of Fred Hutchinson Cancer Research Center in Seattle, and colleagues. “The ability to directly modify a highly enriched CD34+CD90+ cell population to accomplish both short and long-term engraftment would substantially reduce the typically large and expensive amount of vectors or editing agents needed for the genetic engineering of HSCs.”

Recovery after HSC transplantation has been believed for decades to be biphasic, with short-term progenitors driving recovery for the first 6-9 months and multipotent HSCs providing long-term repopulation. Data from human gene therapy trials have used retroviral integration site analysis to track gene-marked cells seeming to confirm this biphasic model.

The investigators performed high-density sampling for retroviral integration site analysis in 2 nonhuman primates, who were followed for 4.5 years. HSC clone signatures were collected and HSC contribution was identified in very early samples to determine the onset of HSC contribution and investigate the kinetics of hematopoietic recovery. The contribution of multipotent HSCs clones was detected during neutrophil recovery ­— a finding is in contrast with the current biphasic model. Moreover, multipotent HSCs became the dominant source for mature blood cells in the peripheral blood as early as 50 days post-transplant.

Further, a hematopoietic reconstitution simulation, informed by observed changes in clonal diversity 

, also predicted that hematopoietic recovery is primarily HSC driven and that HSC contribution follows a random pattern. Lastly, 

 experiments in colony-forming cell assays confirmed that HSCs proliferation and differentiation is a random process.

The investigators noted that the observed changes in the clonal diversity during early recovery suggest a random HSC engraftment and differentiation pattern. Early blood production is driven by HSC clones that are not predetermined but randomly contribute to a limited number of lineages before they disappear—a cell population incorrectly identified as lineage-restricted short-term progenitors, they concluded.

1. Radtke S, Enstrom M, Pande D, et al. Short- and Long-Term Hematopoietic Reconstitution After Transplantation is Stem Cell-driven and Stochastic: Implications for Gene Therapy. Presented at: American Society of Gene & Cell Therapy 25th Annual Meeting; May 16-19, 2022; Washington, DC. Accessed May 17, 2022. 

https://annualmeeting.asgct.org/abstracts/abstract-details?abstractId=6705

2. Six E, Guilloux A, Denis A, et al. Clonal tracking in gene therapy patients reveals a diversity of human hematopoietic differentiation programs. 

Articles

Findings from a preclinical study, which are in contrast with the current biphasic recovery model, have implications for both HSC transplantation and gene therapy.

Hematopoietic Recovery After Myeloablative Transplantation May Be Entirely Driven by Hematopoietic Stem Cells

"Another advantage of macrophages is that they're circulating cells and they migrate out of the blood within hours after the infusion. Therefore, the pro-inflammatory cytokines they released are not causing sustained elevation of cytokines in the blood and we don't see severe cytokine release syndrome with our macrophage therapy.”

A phase 1, first-in-human study is evaluating the safety, tolerability and preliminary efficacy of an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR) macrophage therapy, CT-0508 (Carisma Therapeutics) in solid tumors with HER2 overexpression.

An overview of the study was presented at the 

American Society of Gene and Cell Therapy 25th Annual Meeting

, May 16-19, 2022, in Washington, DC.by Yara Abdou, MD, assistant professor of oncology at University of North Carolina School of Medicine.

spoke with Abdou to learn more about CT-0508 and how CAR macrophage therapies address the challenges of traditional CAR T-therapies in solid tumors. She discussed the progress of the first-in-human, phase 1 study in HER2+ solid tumors.

Stay up to date with coverage from ASGCT 2022 by 

Abdou Y, Yuan Y, Ueno NT, et al. A phase 1, first in human (FIH) study of adenovirally transduced autologous macrophages engineered to contain an anti-HER2 chimeric antigen receptor (CAR) in subjects with HER2 overexpressing solid tumors. Presented at: ASGCT 25th Annual Meeting, May 16-19, 2022; Washington DC. Abstract #805

Videos

The assistant professor of oncology at UNC School of Medicine discussed the first-in-human study being conducted with CT-0508. 

Yara Abdou, MD, on the Advantages of CAR Macrophages in Solid Tumors 

A homology-directed repair (HDR)-based approach has improved efficiency and modulation of T-cell receptor (TCR) therapies for the treatment of type 1 diabetes (T1D).

American Society for Gene and Cell Therapy 25th Annual Meeting, May 16-19, 2022

, in Washington DC, by Martina S. Hunt, research scientist, Seattle Children's Hospital.

“Adoptive regulatory T-Cell (Treg) therapies represent a potentially transformative cell-based therapy to promote immune tolerance following stem cell or solid organ transplantation, and in autoimmune diseases including T1D. Key technical hurdles, however, may limit broad clinical application including the rarity of natural Treg, the requirement for antigen-specificity to modulate tissue-specific disorders, and requirement to compete in vivo for engraftment and survival,” Hunt and colleagues, including first author, Peter J. Cook, PhD, research scientist, Seattle Children's Hospital.

Lentiviral Gene Therapy for Infants With X-Linked SCID Helps Build ‘Functional’ Immune System

The investigators developed an approach that simultaneously induces constitutive expression of FOXP3, a Treg master transcriptional regulator, with the MND promoter to stabilize Treg phenotype and function while replacing the endogenous TCR with a tissue-specific TCR. They also designed donor cassettes with a heterodimeric, chemically-induced signaling complex (CISC) to mimic IL-2 signaling in response to an exogenous dimerizer.

Hunt and colleagues used this approach to generate engineered Treg (EngTregs) products for the prevention and/or treatment of T1D with a TCR specific for the islet antigen IGRP. They observed initial FOXP3/TRAC dual-editing rates ranging from 2% to 15% in healthy donor derived CD4+ T cells with use of CRISPR-based tools. They confirmed positive selection for on-target editing events and negative selection for low-frequency chromosomal translocations following expansion and enrichment.

These cells continued to demonstrate a robust Treg immunophenotype after cryopreservation as well as a secretome switch from pro-inflammatory to immunosuppressive cytokines. Altogether, the investigators concluded that these dual-edited Ag-specific EngTregs had strong suppression activity toward activation and proliferation of effector T cells (Teff) cells, including those expressing a different islet TCR, which demonstrates their significant ability for bystander suppression.

“Dual-edited, Ag-specific, CISC expressing EngTregs represent a promising cell-based therapy for T1D simultaneously predicted to mediate tissue-specific bystander suppression and CISC-mediated enhanced in vivo survival and engraftment. Finally, this dual-editing platform is readily adaptable for use with alternative TCR (or CAR) cassettes designed to target a range of tissue-specific autoimmune or inflammatory diseases,” Cook and colleagues wrote.

Cook PJ, Hunt MS, Yang SJ, et al. Generation of islet-specific engineered regulatory T cells (EngTregs) for immune tolerance induction in type 1 diabetes using a novel dual-editing strategy. Presented at: ASGCT 25th Annual Meeting, May 16-19, 2022; Washington DC. Abstract #810

Investigators from Seattle Children’s Hospital found that engineered T-cell regulatory products demonstrated significant suppression of effector T cells. 

Homology-Directed Repair-Developed TCR Therapy Shows Potential in Type 1 Diabetes 

“The fact that we're getting across the blood brain barrier through the direct injection into cerebrospinal fluid and brain has helped us to, for the first time, show enzyme expression and reduction of gangliosides in the brain... so that's been a milestone."

AXO-AAV-GM2 gene therapy delivered via a combination of bithalamic, intra-cisterna magna (ICM) and intrathecal (IT) methods has been found to be well-tolerated with MRI evidence of accurate bithalamic targeting in all 5 participants with GM2 gangliosidosis dosed so far.

These data, from a phase 1/2 trial (NCT04669535) sponsored by Sio Gene Therapies and conducted at Massachusetts General Hospital and Worcester General Hospital, were presented at the

 2022 American Society of Gene and Cell Therapy Annual Meeting

, May 16-19, 2022, in Washington DC, by Florian Eichler, MD, assistant professor of neurology, Harvard Medical School, and director, leukodystrophy service, Massachusetts General Hospital.

spoke with Eichler during the meeting to learn more about the advantages of the AXO-AAV-GM2 gene therapy and the challenges in treating GM2 gangliosidosis. He also discussed specific advantages of the novel combined delivery approach.

Eichelr F, Flotte T, Andonian H, et al. AXO-AAV-GM2 gene therapy for infantile- and juvenile-onset GM2 gangliosidosis: Preliminary results from an ongoing phase 1/2 trial. Presented at: ASGCT 25th Annual Meeting, May 16-19, 2022, Washington DC.

The director of the leukodystrophy service at Mass Gen discussed the phase 1/2 study of AXO-AAV-GM2. 

Florian Eichler, MD, on How Novel Gene Therapy Delivery Improved Targeting in GM2 Gangliosidosis

Results from the phase 3 HOPE-B trial (NCT03569891) were first shared in December 2021, when it was announced that investigational adeno-associated virus 5 (AAV5) gene therapy etranacogene dezaparvovec (uniQure; CSL Behring) had met its primary end point of reduction in annualized bleeding rate (ABR) post-treatment compared with baseline Factor IX (FIX) prophylactic therapy in patients with hemophilia B (HB).

American Society of Gene & Cell Therapy 25th Annual Meeting

 (ASGCT), held in Washington, DC, and virtually May 16-19, 2022, investigators are sharing their final analysis, in which the therapy continues to produce stable hemostatic correction and improvements in hemophilia-related quality of life via a one-time dose.

HOPE-B, an open-label, single-arm study, was designed to evaluate the safety and efficacy of etranacogene dezaparvovec. A total of 54 adult patients with HB and a baseline FIX of 

 2% were treated (n = 33 with pre-existing AAV5 neutralizing antibodies (NAbs), n = 21 without pre-existing AAV5 neutralizing antibodies). All participants underwent a 

 6-month lead in period of FIX prophylaxis before receipt of 2x1013 gc/kg etranacogene dezaparvovec.

Investigators assessed health-related quality of life (HRQoL) via a hemophilia-specific Hem-A-QoL instrument over the course of the lead-in period and at month 6 and month 12 after treatment and considered a one-sided 

-value ≤ .025 for the post-treatment lead-in period to be statistically significant.

A total of 37 participants experienced 92 adverse events (AEs) that were considered to be related to treatment. However, more than 80% of them (74, 80.4%) were mild in nature. One patient experienced a serious AE, hepatocellular carcinoma, but it was found to be unrelated to the vector.

At the 6-month mark post-treatment, mean FIX activity was 39.0 IU/dL (standard deviation ±18.7; minimum 8.2, maximum 97.1). A year later, at the 18-month mark, it was 36.9 IU/dL (±21.4; 4.5, 122.9). Among participants who received a full dose of etranacogene dezaparvovec, FIX prophylaxis was discontinued by all whose AAV5 NAbs were undetected or who had pre-dosing NAbs up to a titer of 678. This constituted an overall 97% reduction in mean unadjusted annualized FIX consumption.

The 52-week adjusted ABR for all bleeds was reduced 64% (

 = .0002), from 4.19 during the ≥ 6-month lead-in period to 1.51 during months 7-18. This was enough to establish statistical superiority over FIX prophylaxis and achieve the study’s primary end point.

“Following a single dose of etranacogene dezaparvovec, participants experienced a stable and durable increase in mean FIX activity into the near-normal range at 18 months and hemostatic protection, achieving the HOPE-B primary efficacy end point,” investigators concluded.

Participants reported significant improvements across a host of HRQoL domains, resulting in an increased total score overall (LS means -5.50; 

< .0001; percentage improvement 21.5%). Notably, trial participants reported percentage improvements of 45.7% and 59.0% in Feelings (current emotions associated with having hemophilia) and Treatment (burden of hemophilia treatments), respectively, between the lead-in period and the post-gene therapy treatment period.

Etranacogene dezaparvovec was previously granted breakthrough therapy designation by the US Food and Drug Administration and access to Priority Medicine (PRIME) regulatory initiative by the European Medicines Agency (EMA). uniQure developed the gene therapy before entering into a Commercialization and License Agreement with CSL Behring in June 2020 for exclusive global rights to the therapy. It was announced in March that the EMA had begun reviewing a Marketing Authorization Application submitted by CSL Behring for etranacogene dezaparvovec in hemophilia.

“As the first gene therapy candidate for hemophilia B, this pivotal regulatory milestone brings CSL Behring one step closer to delivering on the promise of gene therapy for the bleeding disorders community,” Emmanuelle Lecomte Brisset, head of Global Regulatory Affairs at CSL Behring, said in a statement. “We look forward to working with regulatory authorities to bring the transformative potential of gene therapy to people living with this debilitating, life-long condition.”

Johnson, V. Hemophilia B gene therapy meets primary end points in HOPE-B trial. CGTL website. Published December 9, 2021. Accessed May 12, 2022. 

https://www.cgtlive.com/view/hemophilia-b-gene-therapy-meets-primary-end-points-hope-b-trial

Pipe S, Leebeek F, Recht M, et al. Stable hemostatic correction and improved hemophilia-related quality of life: final analysis from the pivotal phase 3 hope-b trial of etranacogene dezaparvovec. Presented at: American Society of Gene & Cell Therapy 25th Annual Meeting; May 16-19, 2022; Washington, DC. Accessed May 12, 2022. 

https://annualmeeting.asgct.org/abstracts/abstract-details?abstractId=3785

European Medicines Agency commences review of novel gene therapy candidate etranacogene dezaparvovec for people with Hemophilia B. News release. CSL Behring. March 28, 2022. 

https://www.cslbehring.com/newsroom/2022/ema-commences-review-of-novel-gene-therapy-candidate-etranadez-for-people-with-hemophilia-b

The trial met its primary efficacy end point, and participants reported improvements in quality-of-life factors such as outlook on the future and feelings about having hemophilia.

Final Analysis of Phase 3 HOPE-B Trial: Durability of Etranacogene Dezaparvovec and QoL Improvements

Gene therapy has been explored in the past for X-linked severe combined immunodeficiency (XSCID), a rare inherited disorder resulting in a mutation of the 

 gene that encodes a protein that is vital to the development of immune cells, specifically T and natural killer (NK) cells. However, early gene therapy trials successfully restored only the T cells and led to some patients developing iatrogenic leukemia. The traditional treatment option of bone marrow transplant is limited to patients who have a matched donor sibling.

Now, investigators are evaluating a new approach that relies on a lentiviral (LV) vector along with reduced exposure busulfan in newly diagnosed infants with XSCID. Interim results, which include median follow-up of 2.4 years, from the ongoing phase 1/2 trial (NCT01512888) were presented at the 

 (ASGCT), held in Washington, DC, and virtually May 16-19, 2022.

The goal of the study is to assess whether the new method of transferring a normal copy of the common γ-chain gene into the participant's bone marrow stem cells is safe and results in the patient developing a normal immune system.

Primary outcome measures include the number of patients with adequate neutrophil count recovery after busulfan conditioning, number of patients experiencing no directly related grade 4 or greater adverse event (AE), reconstitution with transduced cells defined as detection of vector-marked peripheral blood cells by real time PCR at or above 0.02% VCN [vector copy number ] in total white blood cell count, and number of patients with treatment failure. Other outcomes include number of NK cells, vector copy number by location of vector-integration sites in sorted blood cells,and overall survival, among others.

At ASGCT, investigators reported what they believe to be the longest follow-up data (median, 2.4 years, range: 1.4 months–5.4 years) on the largest cohort of infants with XSCID treated with LV gene therapy. A total of 23 infants with XSCID received the treatment, at a median age of 3 months (range: 2.4–13.8). All patients received busulfan targeted to a cumulative area-under-the-curve (cAUC) of 22 mg*hr/L prior to receiving the infusion of transduced autologous bone marrow CD34+ cells (median vector copy number of 0.81/cell [range: 0.16-1.81], and median CD34+ cell dose of 9.61x106/kg [range 4.4- 18.95]).

According to investigators, all patients are alive and all 20 patients with a follow-up > 4 months have “recovered from pre-existing infections, are off protective isolation and prophylactic antimicrobials, and have normal growth velocity.”

Follow-up data > 6 months was available for 18 patients. Of the 18, 17 exhibited robust immune reconstitution (median CD3+ 2,545/uL [range: 922-4,321], CD4+ 1568/uL [range: 436-3,556], CD4+/CCR7+/CD45RO-/ 1416/uL [range: 298-3,307]). All patients experienced substantial multilineage engraftment that sustained over time, according to VCN analysis in T, B, NK, and myeloid cells separated from peripheral blood.

“In these 17 patients, T cells matured appropriately as assessed by normal T cell receptor excision circles and TCRvβ repertoire diversity and were functional as judged by phytohemagglutinin activation,” investigators reported. “Immunoglobulin replacement has been discontinued in 15 patients, and 12 have been immunized, with 2 more begun immunizations.”

Encouraged by these interim results, investigators hope to further explore LV gene therapy as an alternative treatment modality for infants with XSCID.

“LV gene therapy for XSCID using low dose busulfan conditioning has been well tolerated and results in the development of a durable, functional immune system without evidence of malignant transformation,” the research team concluded. “Thus, our approach presents a promising alternative to current treatment modalities.”

Mamcarz E, Zhou S, Lockey T, et al. Lentiviralgene therapy with low dose busulfan for infants with x-linked severe combined immune deficiency (xscid) results in the development of a normal and sustained immune system: interim results of an ongoing phase I/II clinical study. Presented at: American Society of Gene & Cell Therapy 25th Annual Meeting; May 16-19, 2022; Washington, DC. Accessed May 11, 2022. 

The interim study results presented at ASGCT 2022 likely represent the longest follow-up data to date for the largest cohort of infants with XSCID treated with lentiviral vector gene therapy.