Preclinical Development of Autologous Hematopoietic Stem Cell-Based Gene Therapy for Immune Deficiencies: A Journey from Mouse Cage to Bed Side

Abstract

Recent clinical trials using patient’s own corrected hematopoietic stem cells (HSCs), such as for primary immunodeficiencies (Adenosine deaminase (ADA) deficiency, X-linked Severe Combined Immunodeficiency (SCID), X-linked chronic granulomatous disease (CGD), Wiskott–Aldrich Syndrome (WAS)), have yielded promising results in the clinic; endorsing gene therapy to become standard therapy for a number of diseases. However, the journey to achieve such a successful therapy is not easy, and several challenges have to be overcome. In this review, we will address several different challenges in the development of gene therapy for immune deficiencies using our own experience with Recombinase-activating gene 1 (RAG1) SCID as an example. We will discuss product development (targeting of the therapeutic cells and choice of a suitable vector and delivery method), the proof-of-concept (in vitro and in vivo efficacy, toxicology, and safety), and the final release steps to the clinic (scaling up, good manufacturing practice (GMP) procedures/protocols and regulatory hurdles).

The recombinase activating genes: architects of immune diversity during lymphocyte development

“…Indirect effects of recombination are easier to detect, such as lymphocyte development. Using flow cytometry, one can follow the developmental stages of lymphocytes and observe hindrances or a complete arrest of development when RAG is mutated or disrupted ( 108 , 109 ). Recombination activity can be indirectly measured by (q)PCR of the signal joints from in vitro cultures.…”

“…However, measuring RAG activity directly in vivo is very challenging and has yet to be accomplished. Indirect measuring of the recombination activity is available in the form of the TCR excision circle (TREC) assay, following lymphocyte development in the bone marrow, thymus and peripheral blood through flow cytometry, repertoire analysis, and serum Ig quantification ( 108 ). These methods can be applied, for example, on mice, where RAG-deficient mice are available and serve as good negative controls and targets for RAG gene therapy ( 108 , 112 ).…”

“…We first reported successful LV preclinical work with the spleen focus-forming virus (SFFV) promotor, which was later replaced by the more clinically acceptable myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted (MND) promotor, which has been used before in clinical trials for gene therapy ( 109 , 112 , 197 ). Since then, preclinical studies on lentiviral-based gene therapy for RAG1-SCID has advanced to a phase I/II clinical trial (NCT04797260) in 2021 ( 108 , 109 , 112 , 198 ). These preclinical murine studies used RAG1 deficient mice and performed gene therapy with a lentiviral SIN vector containing codon optimised RAG1 ( coRAG1 ).…”

“…Recent work using HSC based gene therapy for cerebral adreno- leukodystrophy (CALD) using a similar MND promoter has showed 3 cases of myelodysplastic syndrome out of 70 patients treated showing the risk of insertional mutagenesis that is associated with this kind of therapy ( 197 ). For RAG1 LV gene therapy we tested 7 different promoters as high RAG1 expression is required to restore T cell development ( 108 , 112 , 198 , 200 ). In safety assays, the MND-coRAG1 vector has not shown signs of insertional mutagenesis ( 112 , 200 ).…”

Cell-Based Drug Delivery Platforms

“…The work reviews aspects such as product development based on in vivo and ex vivo therapies, depending on target cells of interest, vector designs, ex vivo manipulation and transduction efficiency and the use of models to test the efficacy of gene therapy such as in vitro models or animal models. The safety and toxicology assessment and the scaling-up under good manufacturing practices (GMP) of gene therapy to obtain a product suitable for clinical use are also discussed in the manuscript [ 3 ].…”