New frontiers in the therapy of primary immunodeficiency: From gene addition to gene editing - PubMed (original) (raw)

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New frontiers in the therapy of primary immunodeficiency: From gene addition to gene editing

Donald B Kohn et al. J Allergy Clin Immunol. 2017 Mar.

Abstract

The most severe primary immune deficiency diseases (PIDs) have been successfully treated with allogeneic hematopoietic stem cell transplantation for more than 4 decades. However, such transplantations have the best outcomes when there is a well-matched donor available because immune complications, such as graft-versus-host disease, are greater without a matched sibling donor. Gene therapy has been developed as a method to perform autologous transplantations of a patient's own stem cells that are genetically corrected. Through an iterative bench-to-bedside-and-back process, methods to efficiently add new copies of the relevant gene to hematopoietic stem cells have led to safe and effective treatments for several PIDs, including forms of severe combined immune deficiency, Wiskott-Aldrich syndrome, and chronic granulomatous disease. New methods for gene editing might allow additional PIDs to be treated by gene therapy because they will allow the endogenous gene to be repaired and expressed under its native regulatory elements, which are essential for genes involved in cell processes of signaling, activation, and proliferation. Gene therapy is providing exciting new treatment options for patients with PIDs, and advances are sure to continue.

Keywords: CRISPR/Cas9; Hematopoietic stem cell transplantation; gammaretroviral vector; gene editing; lentiviral vector; site-specific endonuclease; zinc finger nuclease.

Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1

Clinical schema of gene therapy for PID.

Figure 2

Figure 2. Gene delivery vectors used for gene therapy of PID

Upper: Gammaretroviral vectors have viral long terminal repeats (LTR) at each end with viral enhancers and promoters that drive transcription and termination/polyadenylation of a normal copy of the relevant human gene involved in the inherited PID. The messenger RNA produced from the vector is shown as a red arrow including the polyA tail. Lower: Self-inactivating (“SIN”) lentiviral vector has LTR with the enhancers deleted (Δ) and an internal promoter (Prom) to drive transcription of the human PID gene. In current clinical trials, lentiviral vectors being used for ADA SCID and XSCID are using the human Elongation Factor Alpha-1 gene short promoter (EFS) to drive the ADA and IL2Rg genes, respectively, the WASP gene endogenous promoter to drive a WASP gene, and a chimeric myeloid promoter to drive the gp91phox gene for XCGD.

Figure 3

Figure 3. Gene editing using site-specific endonucleases

Site-specific endonucleases (ZFNs, TALENs, or CRISPR/Cas9) can be used to create targeted double stranded DNA breaks. These DNA breaks can be repaired by non-homologous end joining, which introduces small insertions and deletions and can be used for gene knockout strategies, as is employed in the CCR5 gene therapy trials for HIV. If a corrective donor DNA sequence is also provided to the cell, it can be used for homology-directed repair of the double-stranded DNA break, incorporating the therapeutic gene sequence and maintaining its expression under its endogenous regulatory elements.

Figure 4

Figure 4. Site-specific Gene Editing of Autologous Hematopoietic Stem Cells for Gene Therapy of PID

Autologous hematopoietic stem cells (HSC) can be obtained from bone marrow (BM), mobilized peripheral blood stem cells (PBSC) or umbilical cord blood (CB). Usually the HSC are enriched using CD34 immunoselection. The HSC are pre-stimulated to improve gene modification by culture for 24-48 hours with a combination of recombinant hematopoietic growth factors, such as ckit ligand/flt-3 ligand and thrombopoietin (S/F/T). The cells are then treated with electroporation with the site-specific endonuclease (zinc finger nuclease, homing endonuclease, TALEN or CRISPR), to introduce a double stranded break at the target gene, delivered as either in vitro transcribed messenger RNA (mRNA) or pre-formed ribonucleoprotein (RNP) containing the Cas9 protein and a short-guide RNA for CRISPR-mediated processes. The homologous donor containing the corrective sequences may be provided either as an oligonucleotide (Oligo) that is co-electroporated with the endonuclease or via a viral vector (e.g. adeno-associated virus {AAV} or integrase defective lentivirus {IDLV}). Following electroporation, the gene-modified HSC are formulated for intravenous administration and may either be infused into the patient fresh or after cryopreservation and thawing.

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