Translating Gene Therapy to the Clinic

Techniques and Approaches

Jeffrey Laurence, Michael Franklin

• Publisher: Academic Press
• Year: 2014
• Print ISBN: 9780128005637
• eBook ISBN: 9780128005644
• Topic: Scienze biologiche
• Subtopic: Oncologia

Translating Gene Therapy to the Clinic, edited by Dr. Jeffrey Laurence and Michael Franklin, follows the recent, much-lauded special issue of Translational Research in emphasizing clinical milestones and critical barriers to further progress in the clinic. This comprehensive text provides a background for understanding the techniques involved in human gene therapy trials, and expands upon the disease-specific situations in which these new approaches currently have the greatest therapeutic application or potential, and those areas most in need of future research. It emphasizes methods, tools, and experimental approaches used by leaders in the field of translational gene therapy. The book promotes cross-disciplinary communication between the sub-specialties of medicine, and remains unified in theme. - Presents impactful and widely supported research across the spectrum of science, method, implementation and clinical application - Offers disease-based coverage from expert clinician-scientists, covering everything from arthritis to congestive heart failure, as it details specific progress and barriers for current translational use - Provides key background information from immune response through genome engineering and gene transfer, relevant information for practicing clinicians contemplating enrolling patients in gene therapy trials

Chapter 1

Translating Genome Engineering to Survival

Jakub Tolar Stem Cell Institute, Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, MN, USA

Abstract

An incredible number and diversity of genetic diseases afflict humans. Despite the best efforts of medicine to reduce the suffering these diseases cause, curing them has remained out of reach. We are now at the point in time where genome engineering is stunningly close to being able to repair a genetic error and restore gene function. This volume outlines important examples of the tools and practice of gene therapy today. This incredible capability remains at the edges of existing science in new and predictably controversial territory. Similar to any other medical intervention, gene therapy is not devoid of risks. Understanding and controlling these risks while moving these technologies into the practice of medicine remain the goals for translational research. The chapters you are about to read demonstrate that fluency across basic science and clinical practice is possible and necessary for the robust application of gene therapy to improved survival.

Keywords

Blood and marrow transplantation; DNA structure; Eusocial team; Gene editing; Gene therapy; Immunological tolerance; Nuclear transfer; Selfish transgene; Somatic mosaicism; Stem cell therapy

“Daring ideas are like chessmen moved forward; they may be beaten, but they may start a winning game.”

Goethe

list of Abbreviations

DNA

Deoxyribonucleic acid

HCT

Hematopoietic cell transplantation

iPSCs

Induced pluripotent stem cells

mRNA

Messenger ribonucleic acid

SCID

X-linked severe combined immunodeficiency

CRISPR

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system

1.1. Origins

The process of research and its clinical translation is not just driven by doctors and scientists; they are driven by the patients themselves. In the boundaries between science and society, there is a subset of individuals with genetic disorders for whom simply living life is inherently unsafe. These patients and their families frequently view risks of therapy very differently than healthy people do. Probabilities of success or failure that would be unthinkable for a healthy person can be acceptable to those with debilitating or fatal diseases. These extreme situations, in which the possibility of benefit outweighs the significant risk, are fortunately rare but critical to clinical translation. Human suffering is the same whether it is caused by a rare or a widespread disease, but the attention of experts and commitment of society may differ between the two conditions. Because of this disparity, it is important to stress that gene therapy approaches will apply equally to the orphan diseases in which they are pioneered and to the diseases and injuries that cause much of the morbidity and mortality in the world.

Gene therapy has been on the horizon for over 30 years. Encouraged by its early promise, patients and their despairing families have had their hopes raised again and again only to be repeatedly disappointed. However, in 2014, evidence of clear success in selected diseases and the application of safer technologies have added to the intellectual mass needed to forward the process of bringing gene therapy to the clinic, thus rendering countless grim disorders treatable and ultimately curable.

The concept of gene therapy for genetic disorders has its origins in Gregor Mendel’s theory that cells contain small units that are messengers of inherited characteristics¹ and Erwin Schrödinger’s insight that genes are blueprints of cellular function.² Gene therapy is one of the most appealing theories in biomedicine because it is aimed at the cause rather than the symptoms of the disease. However, the ability to harness the potential of gene transfer or DNA repair to correct a specific genomic lesion has simultaneously been one of the most daunting concepts to put into practice, although such “corrective” mutations can occur spontaneously in the setting of somatic cell mosaicism.

Mosaicism, the naturally occurring, spontaneous restoration of function in revertant cells, has been reported in patients with several genetic diseases: the skin disease epidermolysis bullosa; the metabolic disorder hereditary tyrosinemia type I; the bone marrow failure syndromes Fanconi anemia and dyskeratosis congenita; and the primary immunodeficiencies, Wiskott–Aldrich syndrome, Bloom syndrome, X-linked severe combined immunodeficiency (SCID), and adenosine deaminase (ADA)-deficient SCID.^3–14 Therefore, in theory any genetic disease can be spontaneously corrected by a gene conversion, compensatory mutation in cis, or by intragenic crossover between maternal and paternal alleles with two different mutations in the same gene.¹⁵

Despite this knowledge, there clinically are several blocks to applying revertant mosaicism. For example, it remains unclear why in some conditions the self-correction occurs rarely and why in others it may occur repeatedly even in a single individual.^16–18 Furthermore, when self-correction does occur, it may not be clinically meaningful because the revertant event is unpredictable in the molecular mechanism of reversion (and may restore the gene function only partially) and in the location in the gene. Also relevant is the unpredictability of the type of cell in which the reversion occurs, ranging from cells and tissues relevant to the disease phenotype to those that are functionally neutral. It is also currently impossible to predict the type of cell where revertant mosaicism will occur: in stem cells with significant repopulation potential, in progenitor cells—more differentiated but still possessing a variable degree of “stemness”—or in fully differentiated postmitotic cells with limited functional effect beyond the corrected cell.

If revertant mosaicism does not supply the clinical answers we seek, then where do we look next? To see the future, it is useful to first examine the past.

1.2. Synchronicity of Discoveries

The foundations for the practice of stem cell gene therapy today and for optimism about its future effect are three unrelated discoveries made 60 years ago in the 1950s. They are the following:

1. Defining the structure of DNA.¹⁹

2. Inducing immunological tolerance by tissue transplantation.²⁰

3. Creating induced pluripotency by removing the nucleus of an egg and putting in the nucleus of a differentiated cell.²¹

Together, these three discoveries established the science needed for gene therapy to become a reality: from understanding the arrangement of the molecules in DNA, which allowed them to be rearranged; to understanding the mechanisms of immune tolerance, which allowed for cells and tissue to be transplanted; to being able to create a pluripotent cell from a gene-corrected one, which allows these cells to be multiplied and differentiated into different lineages to work in physiologically meaningful ways throughout the body.

1.3. Gene Addition

The discovery of the double-helix structure of DNA by James Watson and Francis Crick is obviously important to gene therapy because it showed that the nucleotides are organized in specific pairs. The understanding of this structure, and of the mechanism by which it divided, opened the new field of genome engineering, in which genes could be cut out of the genome of one cell and spliced into the genome of another as described in this volume by Gersbach. Gene therapy without this recombinant DNA technology is unimaginable.

1.3.1. Genes as Medicine

Of the more than 1800 genetic disorders described in humans, only a small fraction can be treated and even fewer cured. At this time, we are seeing possible cures using gene therapy for sickle cell disease, thalassemia, and ADA deficiency. In a field with so many challenges, these imminent steps into clinical application remind us how important and beneficial successful methods of gene therapy will be. Even with this powerful motivation, there are formidable technological barriers to accomplishing successful gene transfer, such as crossing the cellular membrane, escaping from the endosome, moving through the nuclear membrane, and integrating into the host genome.

The first technological problem is crossing the cellular membrane, the divider between the cell and its environment, a protective barrier that is quite selective about what it will let into the cell. Large molecules such as DNA, which might compromise the cell’s existing DNA, are kept outside. Should the DNA cross the membrane, it must then encounter another cellular defense, the endosome, where material from outside the cell is either broken down into harmless components to be expelled or encapsulated and sent forward for use in the cell. Should the first two obstacles be surmounted, the third and most difficult remains: how to get the cell’s genome, neatly and strictly paired and sequenced, to break open and accept foreign DNA.

1.3.2. Viral Gene Therapy and the “Selfish” Transgene²²

Many of these biological hurdles are surmounted by adapting viral vectors, which are already evolutionarily perfected to penetrate a cell and transfer DNA, for use in gene therapy.²³ This method has its own risks: reactivation of the original virus, formation of tumors or other cancers, and immune reaction to the viral components. The issues of immunity are discussed by Mingozzi in another chapter in...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• Preface
• About the Editors
• Contributors
• Chapter 1. Translating Genome Engineering to Survival
• Chapter 2. Pluripotent Stem Cells and Gene Therapy
• Chapter 3. Genome Engineering for Therapeutic Applications
• Chapter 4. Immune System Obstacles to In vivo Gene Transfer with Adeno-Associated Virus Vectors
• Chapter 5. Risks of Insertional Mutagenesis by DNA Transposons in Cancer Gene Therapy
• Chapter 6. Arthritis Gene Therapy: A Brief History and Perspective
• Chapter 7. Type 1 Diabetes Mellitus: Immune Modulation as a Prerequisite for Successful Gene Therapy Strategies
• Chapter 8. Gene Therapy for Diabetes
• Chapter 9. Gene Therapy for Neurological Diseases
• Chapter 10. Genetic and Cell-Mediated Therapies for Duchenne Muscular Dystrophy
• Chapter 11. Gene Therapy for Retinal Disease
• Chapter 12. Gene Therapy for Hemoglobinopathies: Progress and Challenges
• Chapter 13. Hemophilia Gene Therapy
• Chapter 14. Gene Transfer for Clinical Congestive Heart Failure
• Chapter 15. Gene Therapy for the Prevention of Vein Graft Disease
• Chapter 16. Gene Therapy in Cystic Fibrosis
• Chapter 17. Genetic Engineering of Oncolytic Viruses for Cancer Therapy
• Chapter 18. T Cell-Based Gene Therapy of Cancer
• Chapter 19. Current Status of Gene Therapy for Brain Tumors
• Index