Immunological barriers to haematopoietic stem cell gene therapy

Abstract

Cell and gene therapies using haematopoietic stem cells (HSCs) epitomize the transformative potential of regenerative medicine. Recent clinical successes for gene therapies involving autologous HSC transplantation (HSCT) demonstrate the potential of genetic engineering in this stem cell type for curing disease. With recent advances in CRISPR gene-editing technologies, methodologies for the ex vivo expansion of HSCs and non-genotoxic conditioning protocols, the range of clinical indications for HSC-based gene therapies is expected to significantly expand. However, substantial immunological challenges need to be overcome. These include pre-existing immunity to gene-therapy reagents, immune responses to neoantigens introduced into HSCs by genetic engineering, and unique challenges associated with next-generation and off-the-shelf HSC products. By synthesizing these factors in this Review, we hope to encourage more research to address the immunological issues associated with current and next-generation HSC-based gene therapies to help realize the full potential of this field.

Fig. 1. Haematopoietic stem cell gene therapy.

a–d | Current paradigms for gene therapy of haematopoietic stem cells (HSCs) follow a four-step process: isolation of haematopoietic stem and progenitor cells (HSPCs) from the patient, which contain a population of long-term HSCs (panel a); their ex vivo genetic engineering (for example, using retroviral transduction or CRISPR-based platforms) (panel b); genotoxic conditioning of the patient (typically using the chemotherapeutic busulfan) to create space for transplanted HSCs to engraft (panel c); and transplantation of genetically modified HSPCs back into the patient (panel d). Gene correction and transplantation of multipotent self-renewing HSCs result in the stable reconstitution of a healthy haematopoietic system within the patient.

Fig. 2. Major landmarks in haematopoietic stem cell transplantation and gene therapy.

Timeline highlighting major developments in the fields of haematopoietic stem cell (HSC) transplantation (HSCT) and gene therapy, including both major successes and major clinical challenges arising from immune responses against gene-therapy reagents^,,,,,–. ADA–SCID, adenosine deaminase deficiency–severe combined immunodeficiency.

Fig. 3. Genetic engineering platforms for haematopoietic stem cell gene therapy.

Several platforms have been developed that can be used to engineer the genome of haematopoietic stem cells (HSCs). a | Lentiviral vectors allow for semi-random insertion of transgenes into the genome. b | Site-specific nucleases such as zinc finger nucleases (not shown) and the CRISPR–Cas9 system can mutate regions of the genome through creation of a double-strand break in DNA and its repair through the non-homologous end joining pathway, which creates insertions and/or deletions. In the CRISPR–Cas9 system, a single guide RNA (sgRNA) guides Cas9 to create a double-strand break at a specific target DNA sequence. c | Methods based on homology directed repair rely on the creation of a double-strand break in the genome using a site-specific nuclease (as shown in part b), followed by homology directed repair of the double-strand break using an exogenously supplied DNA donor, from an adeno-associated virus (AAV) vector or a single-stranded oligodeoxynucleotide (ssODN), that has homology to the break site. d | Next-generation gene-editing platforms (such as base editors and prime editing) allow for manipulation of the genome without use of a double-strand break. Cytidine base editors (shown) consist of a catalytically dead Cas9 fused to cytidine deaminase that is guided to the sequence of interest by sgRNA. Cytidine deamination (C → U) followed by mismatch repair can convert a G:C base pair to an A:T base pair. Adenosine base editors (not shown) convert an A:T base pair to a G:C base pair. Prime editors consist of a catalytically impaired (nickase) Cas9 fused to a reverse transcriptase, and a prime editing guide RNA (pegRNA) that contains a sgRNA sequence and a reverse transcriptase template sequence. Cas9 nickase generates a single-stranded break in target DNA, and reverse transcriptase then reverse transcribes pegRNA from the free 3′ DNA end. This initially generates a branched intermediate with the endogenous DNA strand as a 5′ flap. This is then cleaved by endogenous nuclease activity, and ligation repair incorporates the edit into the genome.

Fig. 4. Immune barriers in haematopoietic stem cell gene therapy.

Gene therapy of haematopoietic stem cells (HSCs) is challenged by various immune barriers of both innate and adaptive immune systems. a | Innate immune pathways that detect gene-therapy reagents include double-stranded DNA (dsDNA)-sensing pathways such as those mediated by Toll-like receptor 9 (TLR9) (not shown) and the cGAS–STING pathway; DNA damage response pathways such as through the kinase ATM and p53, which allow for detection of viral episomal DNA in the nucleus; RNA-sensing pathways such as through TLR3, TLR7, TLR8 and TLR13, or through retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), that allow for detection of synthetic or viral RNA; and pathways that allow for the detection of xenogeneic contaminants that can be present when gene-editing reagents such as Cas9 are produced in xenogeneic hosts, such as lipopolysaccharide (LPS) which can be detected by TLR4. The ability of HSCs to digest proteins via the proteasome and to present peptides from these proteins on MHC class I molecules is also shown. b | The adaptive immune system can also present significant challenges to the genetic engineering of HSCs, either when genetically edited cells are transplanted back into a patient or, potentially, if genetic engineering reagents were delivered to cells in vivo. T cells can recognize cells containing foreign proteins (either neoantigens introduced into HSCs or proteins used to genetically engineer them) through interaction with antigen-presenting MHC class I molecules, which leads to T cell activation and destruction of the genetically modified cell. Antibodies produced by B cells can neutralize viral vectors or gene-therapy proteins present in the bloodstream. AAV, adeno-associated virus; IFNγ, interferon-γ; TCR, T cell receptor; TNF, tumour necrosis factor.