Hematopoietic Stem Cell Gene Therapy: Progress and Lessons Learned

Abstract

The use of allogeneic hematopoietic stem cells (HSCs) to treat genetic blood cell diseases has become a clinical standard but is limited by the availability of suitable matched donors and potential immunologic complications. Gene therapy using autologous HSCs should avoid these limitations and thus may be safer. Progressive improvements in techniques for genetic correction of HSCs, by either vector gene addition or gene editing, are facilitating successful treatments for an increasing number of diseases. We highlight the progress, successes, and remaining challenges toward the development of HSC gene therapies and discuss lessons they provide for the development of future clinical stem cell therapies.

Figure 1. Overview of targets for gene therapy

Hematopoietic stem cells (HSCs) isolated from bone marrow can be modified ex vivo and transferred back to the recipient to produce functional, terminally-differentiated cells. Specific cellular targets and the relevant diseases and genes for gene therapy include the following: HSCs: Fanconi Anemia (FANC A–F). Platelets: Hemophilia A (Factor VIII (F8)); Hemophilia B (Factor IX (F9)); Factor X deficiency (Factor X (F10)); Wiskott-Aldrich Syndrome (Wiskott Aldrich Syndrome Protein (WASP)). Neutrophils: X-linked Chronic Granulomatous Disease (Cytochrome B-245 Beta Chain (CYBB)); Kostmann’s Syndrome (Elastase Neutrophil Expressed (ELANE)). Erythrocytes: Alpha-Thalassemia (Hemoglobin Subunit Alpha (HBA)); Beta-Thalassemia and Sickle Cell Disease (Hemoglobin Subunit Beta (HBB)); Pyruvate Kinase Deficiency (Pyruvate Kinase, Liver and RBC (PKLR)); Diamond-Blackfan Anemia (Ribosomal Protein S19 (RPS19)). Monocytes: X-linked Adrenoleukodystrophy (ATP Binding Cassette Subfamily D Member 1 (ABCD1)); Metachromatic Leukodystrophy (Arylsulfatase A (ARSA)); Gaucher disease (Glucosylceramidase Beta (GBA)); Hunter Syndrome (Iduronate 2-Sulfatase (IDS)); Mucopolysaccharidosis type I (Iduronidase, Alpha-L (IDUA)); Osteopetrosis (T-Cell Immune Regulator 1 (TCIRG1)). B Cells: Adenosine deaminase (ADA)-deficient Severe Combined Immunodeficiency (Adenosine Deaminase (ADA)); X-linked severe combined immunodeficiency (Interleukin 2 Receptor Subunit Gamma (IL2RG)); Wiskott-Aldrich Syndrome (Wiskott Aldrich Syndrome Protein (WASP)); X-linked agammaglobulinemia (Bruton’s Tyrosine Kinase (BTK)). T Cells: Adenosine Deaminase (ADA)-deficient Severe Combined Immunodeficiency (ADA); X-linked severe combined immunodeficiency (IL2RG); Wiskott-Aldrich Syndrome Protein (WASP); X-linked Hyper IgM syndrome (CD40 Ligand (CD40LG)); IPEX Syndrome (Forkhead Box P3 (FOXP3)); Early Onset Inflammatory \Disease (Interleukin 4, 10, 13 (IL-4, 10, 13));Hemophagocytic Lymphohistiocytosis (Perforin 1 (PRF1)); Cancer (Artificial T cell receptors (TCR), Cancer; Chimeric Antigen Receptor (CAR)); Human immunodeficiency virus (C-C Motif Chemokine Receptor 5 (CCR5)).

Figure 2. Autologous hematopoietic stem cell transplantation combined with gene addition or editing

(1) Bone marrow (BM) or mobilized peripheral blood (mPB) cells are collected from the patient (red line represents a disease-causing mutation). Typically, 15–20ml of BM/Kg is an acceptable harvest target. While collecting HSCs by mobilization and apheresis is less invasive than BM aspiration, infants have small blood volumes making leukapheresis challenging. Failure to harvest adequate cell numbers can prevent therapy. (2) Modification of HSCs may reduce stem cell capacity. A back-up cell dose of non-modified cells is apportioned to restore native hematopoiesis in the event of graft failure. (3) CD34+ cells are isolated in a GMP-compliant, closed system. Purification of HSCs may reduce total cell number as CD34+ HSCs represent less than one percent of total cells. Alternatively, a CD34+/CD38− enrichment strategy may be employed to further purify HSCs and lower the amount vector required for modification. CD34+ cells may be pre-stimulated ex vivo for 1–3 days prior to modification, depending on the protocol. (4) Gene modification of HSCs must be permanent so as to be passed down to all progeny. Cells are modified by either a viral vector to add a gene (typically requires high concentration vector), or targeted nucleases with/without a donor template to disrupt, correct, or insert a gene. After ex vivo modification, the cell product undergoes release testing to assess purity, identity, safety, potency (transduction/editing efficiency), and other characteristics. If the modification strategy requires selection of corrected cells, low cell yield may prevent transplantation. (5) Prior to receiving the cell product, the patient undergoes conditioning to “make space” for engraftment of modified HSCs (green check represents successful modification of a disease-causing gene). Modified cells may be reinfused fresh or cryopreserved for delivery at a later time. While high-levels of cytoreductive agents may be toxic, inadequate conditioning may result in poor engraftment.

Figure 3. Summary of Gene Editing Pathways

Double stranded break (DSB) is induced by a targeted nuclease (represented by scissors). DSB ends may or may not be resected (dashed or solid line, respectively),. The ultimate gene editing outcome (light blue boxes on the bottom) depends on several factors: the type of donor template provided (yellow box), the phase of cell cycle (light green box) and the presumed DNA repair proteins available (pink box). Gray boxes indicate the names of the repair mechanisms. It should be noted that the figure illustrates the common pathways described to date, however modification of DNA repair pathways and their utilization for gene editing purposes is an area of active research. (From left to right). A DSB with no end resection and no donor available is likely to result in insertions and deletions (indels) and lead to gene disruption via the non-homologous end joining (NHEJ) pathway. NHEJ may occur in any phase of cell cycle. Exogenously providing a double-stranded donor (dsDonor), which contains nuclease cut sites (scissors) around the gene of interest (green rectangle), may result in homology-independent targeted integration (HITI). The presence of microhomology on opposite strands of DNA around the cut site may result in gene disruption via the microhomology-mediated end joining (MMEJ) pathway. A recently reported method of gene integration, termed precise integration into target chromosome (PITCh), utilizes MMEJ machinery to integrate a gene of interest, which is provided by dsDonor with short homology arms (HA) to the DNA (HA are highlighted in orange and blue). The three pathways on the right are generally only active in S/G2 phases of cell cycle and may be used to correct a single nucleotide mutation in the DNA (represented by a red line). Exogenously providing a dsDonor with long homology arms may lead to either gene integration or gene correction via homologous recombination (HR) mechanism, depending on the length of the donor template. A new type of repair mechanism for gene correction was recently described, termed single stranded template repair (SSTR). Although resulting in the same outcome as HR-mediated gene correction, SSTR is presumed to utilize the Fanconi Anemia (FA) pathway and be RAD-51 independent.