Genome-modified hematopoietic stem and progenitor cell (GM-HSPC) therapies represent a significant frontier in the realm of personalized medicine, holding the promise of targeted interventions for a spectrum of disorders far beyond hematological conditions. Development of these therapies is tied intricately to the rich history of and advancements in hematopoietic stem cell (HSC) grafts, which have served as the cornerstone for our understanding of hematopoiesis and bone-marrow transplantation (1).
The journey toward gene-edited stem cell therapies began with the foundational work of E. Donnall Thomas and colleagues during the 1950s and 1960s. Their studies enabled the refinement of protocols for bone-marrow transplantation and demonstrated regenerative potential of HSC grafts transplanted into recipients, thereby reconstituting their blood and immune systems (2). The 1970s brought a shift toward allogeneic HSC transplants, necessitating further refinements in transplantation techniques and immunosuppression strategies (3).
During the 1980s, significant strides were made in improved conditioning regimens, optimizing the success of HSC grafts. Those regimens involved pretransplant chemotherapy or radiation to create a conducive environment for donor-cell engraftment, which helped improve patient outcomes (4). The understanding about CD34 as a cell-surface antigen and marker for hematopoietic stem and progenitor states was a significant milestone. CD34 serves as a valuable tool for identifying and isolating crucial cell populations (5, 6). Its discovery has played a pivotal role in advancing our understanding of hematopoiesis, improving characterization and manipulation of HSCs for therapeutic applications, including bone-marrow transplantation and gene therapy (7).
Advancements in the 1990s introduced the use of peripheral blood stem cells (PBSCs) as an alternative source for HSC grafts (8). Meanwhile, lower-intensity conditioning regimens using reduced doses of radiation and chemotherapy decreased transplant-related complications and expanded eligible patient populations (9).
We must acknowledge all those contributions both in shaping our understanding of transplantation biology and elucidating the potential for developing new GM-HSPCs. Lessons learned from decades of research, coupled with advancements in gene modification, have paved the way for commercialization of several such therapeutics (Table 1).
Table 1: Gene-modified hematopoietic stem and progenitor cell (GM-HSPC) therapeutics commercially approved by the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA). *later withdrawn.
The first publications describing genetic modifications of mammalian cells appeared in the 1980s, showcasing advances using gammaretroviral vectors (10, 11). Those were followed by a series of successful clinical trials that ultimately led to the first licensed GM-HSPC product in 2016: Strimvelis therapy for treatment of adenosine-deaminase–deficient severe combined immunodeficiency (12, 13). Use of lentiviral vectors (LVVs) represented a significant advancement over that of gamma retroviruses, with the former being able to transduce nondividing cells and thus allow expansion of target cell populations. As demonstrated in studies such as Aiuti et al. in 2009 (14), that breakthrough showcased the potential for improved clinical outcomes across a broader spectrum of conditions.
Notably, the approval of several lentivirus-based therapies — namely Zynteglo, Libmeldy, Skysona, and Lyfgenia treatments (Table 1) — emphasizes the clinical success and widespread acceptance of these advancements.
More recently, the field has undergone a transformative shift with the discovery of new endonuclease technologies, particularly clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) (15). The CRISPR/Cas9 system facilitates precise and targeted genetic modifications, which has ushered in new frontiers in gene therapy. Casgevy (exagamglogene autotemcel) was the first CRISPR-based therapy to achieve commercial status, obtaining regulatory approvals in 2023 from the Medicines and Healthcare Products Regulatory Agency (MHRA) in the United Kingdom and the US Food and Drug Administration (FDA) in the United States, with conditional approval so far from the European Medicines Agency (EMA) (16–18). Realizing the potential of hematopoietic gene therapies requires concerted efforts and careful considerations during their development, with emphasized and stringent quality control (QC) testing to ensure the safety, efficacy, and consistency of these innovative products before they can be administered to patients.
In this two-part review, we focus on high-level analytical development considerations for ex vivo GM-HSPC products derived from primary donor cells. Part 1 addresses in-process and drug product control. Part 2, which will be published in BPI’s July-August featured report, examines concerns for HSPC source materials. Look to other recently published reviews for a broader discussion of chemistry, manufacturing, and controls (CMC) for GM-HSPCs (19, 20) and for development considerations with gene-edited pluripotent stem cells (PSCs) (21). Herein we use the term genome modified in a generic sense to include products that are manufactured by means of viral-vector transduction (typically by LVVs) or CRISPR/Cas9–based genome editing.
In-Process and Drug-Product Analytical Controls
In addition to control of the cellular starting materials and genome modification reagents (see Part 2 in July—August), a comprehensive analytical strategy must be implemented to ensure the adequacy of in-process testing measures as well as release and characterization of final HSPC drug products. Some key considerations are addressed below.
Sampling Plan: A sampling plan must balance patient dosing needs with material availability in the context of limited cellular starting material and little to no opportunity for cellular expansion. The total recovery of CD34+ cells from mobilized donors can vary by tenfold or more and depends on factors such as donor age, disease status, and previous treatment history (9). Excessive expansion of CD34+ cells in vitro has been associated with poor engraftment (22–23). Manufacturing processes therefore tend to be designed for static culture, although some efforts are underway to develop approaches for expanding CD34+ cells in culture while maintaining their function (24–25). Therapeutic doses of CD34+ cells typically are set at a minimum of ~2 × 106 cells/kg. Sponsors developing GM-HSPC products often need to set aside a “rescue” or backup dose of unmanipulated cells. Those would be used in case of manufacturing process or supply-chain failure before dosing, but after patient conditioning begins, or when modified cells fail to engraft in recipients (26–27). A recent review by Li et al. provides more detailed discussion regarding how those limitations can affect available cell numbers (19).
In-Process Testing: In-process testing occurs at nearly every unit operation of GM-HSPC manufacturing processes (Figure 1). Minimally, cell count and viability measurements are assessed to monitor a process and establish its efficiency. Measurement of CD34+ cell predominance — as well as the presence of nontarget cell types — can help ensure appropriate enrichment of the target cells while monitoring depletion of unwanted cell types. Monitoring residual levels of genome-modification reagents also can demonstrate clearance after introduction of those reagents and during the genome-editing unit operation. Early in development, most such measurements will be used for information purposes only. As more experience is gained with a given process, it is expected that many (if not most) of those measurements will become control points. Note that assays used as control points should be qualified or validated.
Figure 1: Standard manufacturing process for genome-modified hematopoietic stem and progenitor cell (GM-HSPC) therapies; sgRNA = single-guide ribonucleic acid.
Release Testing: Table 2 provides a nonexhaustive list of typical attributes that might be included for release of a GM-HSPC product. Not every assay listed will be present on every GM-HSPC release specification. Cell count and viability assays form the cornerstone of nearly all analytical control strategies for CGTs because such assays are heavily used both for in-process monitoring and for defining drug product dosages. The assays are considered to be straightforward, although they do involve many sources of variation, and analysis of cryopreserved products can bring additional performance variability (29–30). Flow-cytometry–based assays commonly are used to evaluate cellular purity and report the percentage of CD34+ cells present. To minimize risks of graft-versus-host disease (GvHD), the percentage of cells expressing the canonical T-cell marker CD3 also might be included within panels for allogeneic products (31–32).
Table 2: Standard release panel for genetically modified hematopoietic stem and progenitor cell (GM-HSPC) therapeutics. Abbreviations: CFU = colony forming unit, ddPCR = droplet digital polymerase chain reaction (Thermo Fisher Scientific), ELISA = enzyme-linked immunosorbent assay, GVHD = graft-versus-host disease, LVV = lentiviral vector, MoA = mechanism of action, USP = United States Pharmacopeia, qPCR = quantitative polymerase chain reaction.
Potency testing is a critical aspect of ensuring the efficacy and biological activity of GM-HSPC products that represents a persistent challenge in their development. Salmikangas et al. noted that “major issues with potency tests were noted in almost 50% of all ATMP MAAs [advanced-therapy medicinal product market-authorisation applications] in the EU [European Union]” (32). The authors cite a statement from Peter Marks’s (director of the FDA’s Center for Biologics Evaluation and Research (CBER)) that “issues related to potency” have been a key delaying factor for many product reviews (33–35).
Potency evaluation is expected to reflect a product’s mechanism(s) of action (MoA) , which may be complex and multileveled; ideally, potency results should correlate with clinical responses (36–37). However, current regulatory guidance provides a degree of flexibility based on a product’s stage of development. For example, a 2011 FDA guidance notes that, for phase 1 and early phase 2 studies, “limited quantitative information on relevant biological attributes may be sufficient”, whereas at later phases reviewers expect potency-testing strategies to “assure that product lots are well defined, biologically active, and consistently manufactured” (37).
A recent update to the potency guidance (currently in draft form) could expand further upon that regulatory flexibility by introducing the concept of a “potency assurance strategy,” whereby a sponsor may “reduce risks to potency by controlling aspects of the manufacturing process that may affect potency” (37). This revised approach could provide sponsors with additional flexibility in how to establish a potency strategy, particularly early in development.
Despite significant regulatory flexibility with respect to a sponsor’s potency strategy, it is critical that GM-HSPC sponsors implement quantitative and fit-for-purpose potency assays and consider a matrixed testing approach to cover multiple aspects relating to MoAs (36). As the draft updated potency guidance notes, release assays should be qualified before initiation of clinical studies to support licensure, and assays for release of licensed products must be validated (37). Early product characterization plays a pivotal role in the identification of both potency-indicating product attributes and the cellular processes that require control for maintaining potency during release testing.
Assessment of the functional capabilities of gene-modified HSCs involves a complex interplay of several factors: cellular viability and ability to engraft, differentiate, and produce desired therapeutic effects (33). Of significant importance is the development of a colony-forming unit (CFU) assay, which provides predictive value for successful hematopoietic engraftment by measuring the potential of HSCs to proliferate and differentiate (38). For GM-HSPCs, assessments of potency also should consider the consequent effects of genomic modification. For products manufactured using viral-vector transduction, early stage potency assessment requires evaluation of both transduction efficiency (assessable by flow cytometry) and vector copy number, which typically is assessed through polymerase chain reaction (PCR)–based methods (39, 40).
For GM-HSPCs, an evaluation of on-target editing efficiency may be a suitable measure of potency early in development, and multiple approaches to doing so have been described (41, 42). Regardless of the type of genetic modification used, the suite of potency assays for a commercial product should evaluate genomic modification functionality and the therapy’s intended MoA (33). Such inclusion can be extremely challenging for HSPCs because, depending on the gene, changes in expression and/or function might not be measurable until cells have differentiated. That can take two to three weeks, making it difficult to implement such testing in a QC-friendly way (33). Thus, the multifaceted nature of potency testing reflects the complexity of GM-HSPC product characterization, requiring a comprehensive and tailored approach to ensuring safety and efficacy for therapeutic applications.
A comprehensive genomic safety assessment is critical to ensuring the safety of GM-HSPC products, with specifics depending on the genome-modification approach applied. For example, although CRISPR/Cas9 is high-precision in nature, unintended genetic events can occur when using the system: e.g., off-target insertions and deletions (INDELs), translocations, and generation of DNA structural variants (43, 45). FDA guidance sets expectations for developers seeking to assess genomic safety (44). As noted, developers should use “multiple methods (e.g., in silico, biochemical, cellular-based assays)” that include at least one genome-wide method to identify candidate off-target sites. Several in silico, biochemical, and cell-based methods have been established for off-target detection (46–48).
Development strategies also should account for the possibility of sequence gaps or mismatches between single-guide RNA (sgRNA) and off-target sites as well as sequence diversity within a cell population (e.g., single-nucleotide polymorphisms, SNPs). Candidate off-target sites that are confirmed through testing of multiple drug-product batches should be assessed for risk, with high levels assigned to sites located in coding regions, associated with a known cellular function, and/or involved in disease (e.g., cancer). Sponsors should consider developing release assays for such high-risk off-target sites. Note that although the conceptual framework for evaluating the potential for translocations and structural variants is similar to that for INDELs, detection of such events often is not possible with methods typically used to identify off-target INDELs (45, 49).
For LVV-based products, the primary genomic safety risk is posed by uncontrolled integration, which can disrupt adjacent genes. To ensure safety in clinical applications, developers must aim for minimal integration copy numbers while maintaining an acceptable level of both transduction efficiency and drug-product potency (50). High posttransduction copy numbers carry the risk of genotoxicity and could increase the likelihood of transgene integration near oncogenes (51). Common techniques used for measuring viral copy numbers integrated into a host genome include quantitative PCR (qPCR) and digital PCR (dPCR). The field is progressing toward standardization of those assays, emphasizing the use of appropriate reference-standard materials to enable accurate comparisons (50, 52).
Characterization Testing: In addition to release assays, developers also should develop a suite of assays to characterize their drug products fully. Such assays could be performed routinely on every manufactured batch or selectively on specific batches, depending on the level of risk identified with a given aspect measured. These assays could include flow-cytometry–based methods for evaluation of HSPC subpopulations and confirmation that a genome-modification process has not affected the distribution of subpopulations unintentionally (39).
GM-HSPC products can contain several residual materials, including nontarget cells (such as T cells, B cells, monocytes, and neutrophils from donor leukapheresis) or residual genome-modification reagents (53). Cell-based residuals can be identified by flow cytometry. Enzyme-linked immunosorbent assays (ELISAs) and PCR-based methods typically are used to detect residual Cas9, sgRNA, or viral vectors.
The most important role for characterization testing might be to provide additional supportive evidence for potency and safety of a drug product. Assays that support drug-product potency are unsuitable for implementation in a QC environment — e.g., those that are only semiquantitative or highly variable in their performance — should be identified and performed for the purpose of characterization instead. Genomic-safety assays also might be performed to characterize drug products, such as when low-risk off-target editing sites are analyzed routinely to evaluate process consistency rather than for release of the drug product. Furthermore, vector–copy-number analyses could be conducted at a single-cell level to enhance understanding of viral integration dynamics with increased granularity (54–55).
Finally, a thorough genomic safety evaluation of LVV-derived products should include analysis of their integration-site profiles within a host genome. Evaluating the specific genomic locations where LVVs integrate is imperative for determining the risks of genotoxicity and potential adverse effects, particularly when integration takes place near oncogenes (56–58). Researchers use a number of molecular techniques such as PCR, next-generation sequencing (NGS), and transposase-based approaches to examine integration sites comprehensively (59). The resulting information is pivotal to optimizing vector design, assessing the safety profiles of gene-therapy vectors, and guiding regulatory assessments for clinical applications (60). The World Health Organization’s (WHO’s) recent establishment of a reference standard for integration-site analysis underscores the global importance of this pivotal aspect of gene therapy (61).
Looking Ahead
Whether manufactured through viral-vector–mediated gene delivery, nuclease-mediated editing, or both, genome modification of HSPCs represents a significant advancement in the potential to cure diseases that otherwise have suboptimal or no currently available treatment options. Building on a substantial therapeutic legacy of HSC transplants, these new therapies are complex to manufacture and require broad and deep analytical support to ensure adequate and consistent product quality (13).
GM-HSPCs face two key analytical challenges. First, the components used to manufacture them are often bespoke and require significant analytical oversight, as will be expanded upon in Part 2 of this review. Second, the broad range of materials used to manufacture a GM-HSPC can include proteins, nucleic acids, viral vectors, and cellular materials. Each of those requires the development of a bespoke analytical approach, including specific analytical tools and methods. Challenges associated with appropriate characterization of GM-HSPC products will increase as the field matures, with the potential addition of in vivo targeting approaches and new gene-editing techniques — such as base and prime editing and gene writing (62–64) — and in vivo targeting approaches (65).
Thus, GM-HSPC sponsors are advised to put significant thought into the development of appropriate analytical control strategies for each of their candidate therapies. That can help maximize the probability of regulatory, technical, clinical, and commercial success, thus enabling each candidate to maximize the likelihood of achieving its therapeutic potential.
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Corresponding author Brent Morse is a principal consultant, and Alicja Fiedorowicz is an analytical consultant in cell and gene therapy at Dark Horse Consulting Group near Boston, MA; [email protected].
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