Drug Development for Gene Therapy E-Book
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- Herausgeber: John Wiley & Sons
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
Drug Development for Gene Therapy Industry-centric perspective on translational and bioanalytical challenges and best practices for gene therapies Drug Development for Gene Therapy focuses on the translational and bioanalytical challenges and best practices for gene therapy modalities, presenting a significant body of data, including information related to safety and efficacy, necessary to advance through the development pipeline into clinical use. The text covers bioanalytical methods and platforms including patient screening assays, different PCR tests, enzyme activity assays, ELISpot, NGS, LC/MS, and immunoassays, with FDA and EMA guidelines on gene therapy safety and efficacy, along with companion diagnostics regulations from US and EU perspectives. The chapters offer an in-depth discussion of the basics and best practices for translational biomarkers, bioanalysis, and developing companion diagnostics / lab tests for gene therapies in the pharma and biopharma industries. To aid in reader comprehension, the text includes clinical examples of relevant therapies in related chapters. Some of the core topics covered include study design, immunogenicity, various bioanalytical methods and their applications, and global regulatory issues. Written by two highly qualified authors with significant experience in the field, Drug Development for Gene Therapy includes information on: * Bioanalytical methods to detect pre-existing antibodies against adeno-associated viruses (AAV) capsids * Detection of cellular immunity and humoral response to viral capsids and transgene proteins, and immunogenicity of gene therapy products * Nonclinical and clinical study considerations and methods for biodistribution and shedding * Quantification of transgene protein expression and biochemical function, and substrate and distal pharmacodynamic biomarker measurements for gene therapy * Detection and quantification of rAAV integration and off-target editing * Current regulatory landscape for gene therapy product development and the role of biomarkers and general regulatory considerations for gene therapy companion diagnostics With comprehensive coverage of the subject, Drug Development for Gene Therapy is a must-have resource for researchers and developers in the areas of pharmaceuticals, biopharmaceuticals, and contract research organizations (CROs), along with professors, researchers, and advanced students in chemistry, biological, biomedical engineering, pharmaceuticals, and medical sciences.
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Table of Contents
Cover
Table of Contents
Title Page
Copyright
List of Contributors
Preface
Section I: Introduction
1 Introduction to AAV‐based
in vivo
Gene Therapy
1.1 Introduction
1.2 Advantages and Disadvantages for AAV
in vivo
1.3 Technology Platforms of AAV‐based
in vivo
Gene Therapy
1.4 AAV Serotypes and Tissue Affinity
1.5 Precision Medicine: Screening and Monitoring Biomarkers, Companion Diagnostics
1.6 Predictions for Scientific and Medical Progress
1.7 Predictions for Market Adoption
1.8 Final Thoughts
References
2 Recent Development in
in vivo
Clinical Gene Therapy Platforms
2.1 Introduction
References
Section II: Translational Biomarkers for Gene Therapy
3 Biomarker and Bioanalytical Readouts for the Development of AAV Gene Therapy
3.1 Introduction
3.2 Pharmacokinetic (PK) and Pharmacodynamic (PD) Biomarkers
3.3 Safety and Monitoring Biomarkers and Readouts
3.4 Predictive and Diagnostic Biomarkers for Study Enrollment and Patient Stratification
3.5 Summary
References
4 Nonclinical and Clinical Study Considerations for Biodistribution, Shedding, and Pharmacokinetics/Pharmacodynamics
4.1 Biodistribution and Viral Shedding
4.2 Pharmacokinetic/Pharmacodynamic (PK/PD) Modeling and Clinical Dose Selection of Gene Therapy
4.3 Summary
References
5 Immunogenicity of AAV Gene Therapy Products
5.1 Innate and Adaptive Immunity Induced by AAV‐Based Gene Therapies
5.2 Preclinical Immunogenicity Risk Assessment
5.3 Clinical Manifestation Associated with Immunogenicity
5.4 Clinical Mitigation Strategy
References
Section III: Bioanalysis for Gene Therapy
6 Bioanalytical Methods to Detect Preexisting and Post‐administration Humoral Immune Responses Against AAV Capsid Proteins
6.1 Introduction
6.2 Considerations for AAV Total Antibody Assays
6.3 Considerations for Cell‐based Transduction Inhibition Assays
References
7 Bioanalytical Methods to Study Biodistribution and Shedding of AAV‐Based Gene Therapy Vectors
7.1 Introduction
7.2 Choice of Platform: qPCR vs. Digital PCR
7.3 Aspects of Method Development
7.4 Back‐Calculation Formulas and Extraction Efficiency Assessments
7.5 Sensitivity Requirements
7.6 Specificity Requirements
7.7 Standard Curve Performance, Colinearity, Precision, and Accuracy
7.8 Selectivity Assessment and Matrix Interference
7.9 Sample Stability Considerations
7.10 Data Reporting Formats, Acceptance Criteria, and Trending
7.11 Immunocapture qPCR: An Ultra‐Sensitive Method to Detect Intact AAV Capsids
References
Note
8 Transgene mRNA Expression Analysis
8.1 Purpose of Measuring Transgene mRNA
8.2 Technologies to Quantify Transgene Expression in Tissues
8.3 Summary
References
9 Quantification of Transgene Protein Expression and Biochemical Function
9.1 Introduction
9.2 Transgene Protein Concentration Determination
9.3 Transgene Protein Activity Determination
9.4 Summary
References
10 Substrate and Distal Pharmacodynamic Biomarker Measurements for Gene Therapy
10.1 Introduction
10.2 Technologies to Quantify Substrate and Distal PD Biomarker
10.3 Summary
References
11 Detection of Cellular Immunity to Viral Capsids and Transgene Proteins
11.1 Introduction
11.2 Methods for the Detection of Cellular Immune Responses
11.3 Validation of Cellular Assays Using PBMC (Example ELISPOT)
References
12 Detection of Humoral Response to Transgene Protein and Gene Editing Reagents
12.1 Pre‐ and Post‐dose Humoral Immunity to Transgene‐expressed Proteins
12.2 Relevance of Analytical Protocols Applied in Determining Immune Response to Protein Therapeutics to the Detection of Anti‐Transgene Protein Responses
12.3 Analysis of Immune Response by Binding and Functional Antibody Assay Protocols
12.4 Comparative Analysis of the Immune Response Evaluation for Transgene Proteins that are Expressed Extracellularly vs. Intracellularly
12.5 Humoral Immune Response to Gene Editing Reagents
References
13 rAAV Integration: Detection and Risk Assessment
13.1 Introduction
13.2 Review of Regulatory Guidance and Discussion Points that Are Raised on AAV Carcinogenesis
13.3 Assessing the Biologic Relevance of AAV Integration Profile
13.4 Conclusion and Future Direction
References
14 Detection and Quantification of Genome Editing Events in Preclinical and Clinical Studies
14.1 Introduction
14.2 Regulatory Guidance on Engineered Nuclease On‐ and Off‐target Assessment
14.3 Strategies and Methodologies to Evaluate On‐target and Off‐target Activities
14.4 Concluding Remarks
References
Section IV: Companion Diagnostic Development for Gene Therapy
15 Introduction to Companion Diagnostics for Gene Therapy
15.1 Introduction to Companion Diagnostics
15.2 Role in Gene Therapy
15.3 Overall Strategy
15.4 Development Process
15.5 Considerations for Commercialization
15.6 Conclusion
References
16 Validation for Gene Therapy Companion Diagnostics
16.1 Introduction
16.2 Development of CTAs for Use in GTx Clinical Trials
16.3 Best Practices for Sample Banking and Consent of Subjects
16.4 Design Considerations
16.5 Bridging Studies
16.6 Commensurate Regulatory Review and Approval of GTx CDx
16.7 Concluding Sections
References
17 Regulatory Considerations for Gene Therapy Companion Diagnostics
17.1 Introduction
17.2 US FDA
17.3 European Union
17.4 Other Regulated Markets
17.5 Development Strategy with the Therapeutic
17.6 Partner Relationship
17.7 Commercial and Post‐Approval Considerations
17.8 Final Word
References
Section V: Regulatory Perspectives on Gene Therapy
18 Current Regulatory Landscape for Gene Therapy Product Development and the Role of Biomarkers
18.1 Introduction
18.2 What is Gene Therapy?
18.3 Biomarkers Defined
18.4 Early Gene Therapy Biomarkers
18.5 Current Expectations for Gene Therapy Biomarkers
18.6 Safety Biomarkers for Gene Therapy Products
18.7 Concluding Remarks
References
Index
End User License Agreement
List of Tables
Chapter 1
Table 1.1 FDA‐approved cellular and gene therapies.
Table 1.2 AAV gene therapy clinical trial status.
Table 1.3 Complete and active AAV gene therapy clinical trial indications....
Chapter 2
Table 2.1 Clinical trials with AAV‐mediated gene replacement therapy or inh...
Table 2.2 Clinical trials with AAV-mediated gene replacement therapy for bra...
Table 2.3 Clinical trials with AAV‐mediated gene replacement therapy for he...
Table 2.4 Clinical trials with AAV‐mediated gene replacement therapy hematol...
Table 2.5 Clinical trials with AAV‐mediated gene replacement therapy for sk...
Chapter 4
Table 4.1 Summary of guidelines, concept papers, and authority consideratio...
Table 4.2 Natural tissue tropism of select AAV serotypes [14].
Table 4.3 Mechanisms of potential adverse events during and after recombina...
Table 4.4 Select examples of recombinant AAV gene therapy by transgene prod...
Table 4.5 Select examples of scaling the total vector genome dose for recom...
Table 4.6 Potential factors leading to loss of transgene product levels and...
Chapter 6
Table 6.1 Theoretical neutralizing capacity of plasma volumes
in vitro
and
Table 6.2 Theoretical relationship between TI titer and percentage dose neu...
Chapter 7
Table 7.1 Characteristics of qPCR and ddPCR assays.
Table 7.2 Example of extraction efficiency assessment during PCR method val...
Table 7.3 Exemplary standard curve, QC, and run acceptance criteria for PCR...
Table 7.4 Exemplary test sample acceptance criteria for PCR.
Chapter 8
Table 8.1 Commonly used instruments for total RNA quality check and quantif...
Table 8.2 Comparative overview of RT‐qPCR vs. RT‐dPCR.
Table 8.3 Currently available digital PCR systems.
Table 8.4 Recommendations for RT‐qPCR/RT‐dPCR method validation for regulate...
Table 8.5 Outlines of RNA‐ISH procedure.
Chapter 9
Table 9.1 Fluorescent background.
Chapter 10
Table 10.1 Biomarker purpose and suggested fit‐for‐purpose validation exten...
Table 10.2 Current spectrum of technologies and platforms for quantitative ...
Table 10.3 Comparison of method validation parameters between fully validat...
Table 10.4 Frequently used reference genes in RT‐qPCR‐based relative quanti...
Table 10.5 Comparison between RT‐qPCR – RNA‐seq and Nanostring.
Chapter 13
Table 13.1 List of studies with rAAV administration in neonatal and adult m...
Table 13.2 List of studies with rAAV administration in large animal species...
Chapter 14
Table 14.1 Targeted approaches to measure short insertions and deletions.
Table 14.2 Technologies to measure large genomic rearrangements.
Table 14.3 Comparison of molecular assays for genome‐wide assessment of gen...
Chapter 16
Table 16.1 FDA guidance documents for industry that provide recommendations...
Table 16.2 Typical studies required for validation and FDA submission of a ...
Chapter 17
Table 17.1 Regulation of companion diagnostics in key markets.
List of Illustrations
Chapter 1
Figure 1.1 Timeline of scientific advances in gene therapy research [1].
Figure 1.2 Creation of recombinant AAV particles.
Figure 1.3 rAAV vector entry and transduction pathway. (1) AAV vectors bind ...
Figure 1.4 Gene editing techniques using double‐stranded breaks. ZFN and TAL...
Chapter 2
Figure 2.1 Target organs for AAV‐mediated gene therapies in clinical develop...
Chapter 3
Figure 3.1 Biomarker categories according to BEST and examples of context of...
Chapter 4
Figure 4.1 Biodistribution and transgene expression pathways of a recombinan...
Figure 4.2 General strategy for selection of recombinant AAV doses in Phase ...
Figure 4.3 Scenarios of transgene product or pharmacodynamic response over t...
Chapter 5
Figure 5.1 AAV‐mediated activation of innate and adaptive phase immune respo...
Chapter 6
Figure 6.1 Principles of antigen capture (a) and bridging (b) assay formats ...
Figure 6.2 Principle of cell‐based AAV transduction inhibition (TI) assays....
Figure 6.3 LOD interpolation at the assay cutpoint set for 50% transduction....
Figure 6.4 Theoretical relationship between TI titer and total AAV dose neut...
Figure 6.5 Theoretical dose neutralization curves were obtained by plotting ...
Chapter 7
Figure 7.1 Optimizing the annealing temperature for thermal cycling in a dup...
Figure 7.2 Exemplary formula to back‐calculate copy numbers of vector genome...
Figure 7.3 Formula to back‐calculate copy numbers of vector genomes per mg t...
Chapter 8
Figure 8.1 The AAV‐mediated GTx may deliver direct transcript from the AAV v...
Figure 8.2 Schematic of RT‐qPCR/RT‐dPCR for gene expression analysis in GTx ...
Chapter 9
Figure 9.1 Michaelis–Menten Equation. “
V
0
” denotes reaction velocity and “[
Figure 9.2 Time course of an enzyme activity expressed in nmol/mL of the mat...
Figure 9.3 Enzyme activity. Lysosomal storage enzyme activity in relation to...
Figure 9.4 Plasma sample processing comparison. Four donors (represented by ...
Figure 9.5 Impact of surfactants. Three donors represented by S1 (open circl...
Figure 9.6 Enzyme activity response. Lysosomal storage enzyme activity in re...
Chapter 10
Figure 10.1 Principle of NanoString nCounter for RNA relative quantification...
Figure 10.2 Guidelines for steps/workflow in practice for quantitation of AA...
Chapter 11
Figure 11.1 Schematic diagram of ELISPOT assay. Ninety‐six well plates are c...
Figure 11.2 Storage temperature of whole blood influences the functionality ...
Figure 11.3 Cryopreserved PBMCs generated from whole blood at different time...
Figure 11.4 Fresh and cryopreserved PBMC perform equally well in recall anti...
Chapter 12
Figure 12.1
ELISA and ECL assay formats for the detection of total antibodie
...
Figure 12.2 ECL assay for the detection of neutralizing antibodies to transg...
Chapter 13
Figure 13.1 Common analysis steps in AAV GTx safety analysis. In light blue ...
Figure 13.2 Library preparation methods for vector integration site analysis...
Chapter 14
Figure 14.1 Molecular outcomes of gene editing events by engineered nuclease...
Figure 14.2
Ex vivo
and
in vivo
genome editing for clinical applications. Le...
Figure 14.3 Workflows for evaluating off‐target activities during genome edi...
Figure 14.4
Techniques to identify genome‐wide off‐target sites. (a) GUIDE‐s
...
Figure 14.5 Targeted approaches to measure short insertions and deletions. (...
Figure 14.6 Technologies to measure large genomic rearrangements. (a) AMP‐se...
Chapter 15
Figure 15.1 Process diagram for development of a companion diagnostic.
Chapter 17
Figure 17.1 Codevelopment of a therapeutic and companion diagnostic from FDA...
Guide
Cover
Table of Contents
Title Page
Copyright
List of Contributors
Preface
Begin Reading
Index
End User License Agreement
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Drug Development for Gene Therapy
Translational Biomarkers, Bioanalysis, and Companion Diagnostics
Edited by
Yanmei Lu
Sangamo Therapeutics
Richmond, California
USA
Boris Gorovits
Gorovits BioSolutions, LLC
Andover, Massachusetts
USA
Copyright © 2024 by John Wiley & Sons, Inc. All rights reserved.
Published by John Wiley & Sons, Inc., Hoboken, New Jersey.Published simultaneously in Canada.
No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per‐copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 750‐8400, fax (978) 750‐4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748‐6011, fax (201) 748‐6008, or online at http://www.wiley.com/go/permission.
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Library of Congress Cataloging‐in‐Publication Data
Names: Lu, Yanmei, 1966‐ editor. | Gorovits, Boris, editor.
Title: Drug development for gene therapy : translational biomarkers, bioanalysis, and companion diagnostics / edited by Yanmei Lu, Boris Gorovits.
Description: Hoboken, New Jersey : John Wiley & Sons, Inc., [2024] | Includes bibliographical references and index.
Identifiers: LCCN 2023049872 (print) | LCCN 2023049873 (ebook) | ISBN 9781119852780 (cloth) | ISBN 9781119852797 (adobe pdf) | ISBN 9781119852803 (epub)
Subjects: MESH: Genetic Therapy–methods | Biomarkers, Pharmacological–analysis | Drug Development–methods
Classification: LCC RB155 (print) | LCC RB155 (ebook) | NLM QU 560 | DDC 616/.042–dc23/eng/20231214
LC record available at https://lccn.loc.gov/2023049872
LC ebook record available at https://lccn.loc.gov/2023049873
Cover Design: Wiley
Cover Image: © Jonathan Knowles/Getty Images
List of Contributors
Editors
Yanmei Lu
Biomarker and BioAnalytical Sciences
Sangamo Therapeutics
Richmond, California
USA
Boris Gorovits
Translational Sciences, Bioanalysis & Biomarkers
Gorovits BioSolutions, LLC
Andover, Massachusetts
USA
Authors
Paul Bartel
Companion Diagnostics
Myriad Genetics, Inc.
Salt Lake City, Utah
USA
Manuela Braun
Bayer AG
Berlin
Germany
George Buchlis
Department of Medicine
University of Pennsylvania
Philadelphia, Pennsylvania
USA
Liching Cao
Biomarker and Bioanalytical Sciences
Sangamo Therapeutics
Richmond, California
USA
Kennon Daniels
Precision for Medicine
Bethesda Metro Center
Maryland
USA
Maurus de la Rosa
Sangamo Therapeutics Allée de la Nertière
Valbonne
France
Robert Dodge
Department of BioMedical Research
Novartis
East Hanover, New Jersey
USA
Mica Elizalde
Regulatory Digital Health
Merck Sharp & Dohme LLC
Rahway, New Jersey
USA
Marina Falaleeva
Preclinical Department
Sangamo Therapeutics
Richmond, California
USA
Raffaele Fronza
ProtaGene CGT GmbH
Heidelberg
Germany
Irene Gil‐Farina
ProtaGene CGT GmbH
Heidelberg
Germany
Jennifer Granger
PharmaDx
ARUP Laboratories
Salt Lake City, Utah
USA
Michael Havert
Gene Therapy Partners, LLC
Arlington, Virginia
USA
Vibha Jawa
Clinical Pharmacology, Pharmacometrics, Disposition and Bioanalysis (CPPDB)
Bristol Myers Squibb
Princeton, New Jersey
USA
Wibke Lembke
Celerion Switzerland AG
Fehraltorf
Switzerland
John Lin
Frontage Laboratories
Exton, Pennsylvania
USA
Hsing‐Yin Liu
Molecular Biology, Johnson and Johnson Innovative Medicine
Janssen Pharmaceuticals
Spring House, Pennsylvania
USA
Kathleen Meyer
Preclinical Department
Sangamo Therapeutics
Richmond, California
USA
John E. Murphy
Arbor Biotechnologies
Cambridge, Massachusetts
USA
Jane Owens
Rare Disease Research Unit
Pfizer Inc.
Cambridge, Massachusetts
USA
Karen L. Richards
Precision for Medicine
Bethesda Metro Center
Maryland
USA
Laura I. Salazar‐Fontana
LAIZ Reg Science Consulting
Lausanne
Switzerland
Oscar Segurado
ASC Therapeutics
Milpitas, California
USA
Russell K. Soon Jr
.
BioMarin Pharmaceutical, Inc.
Novato, California
USA
Kefeng Sun
Quantitative Clinical Pharmacology, Data Sciences Institute
Takeda Development Center Americas
Cambridge, Massachusetts
USA
Magdalena Tary‐Lehmann
CTL‐Contract Laboratory
Cellular Technology Limited
Shaker Heights, Ohio
USA
Shengdar Q. Tsai
Department of Hematology
St Jude Children’s Research Hospital
Memphis, Tennessee
USA
Venkata Vepachedu
Molecular Biology, Johnson and Johnson Innovative Medicine
Janssen Pharmaceuticals
Spring House, Pennsylvania
USA
Christian Vettermann
BioMarin Pharmaceutical, Inc.
Novato, California
USA
Kai Wang
GlaxoSmithKline
Collegeville, Pennsylvania
USA
Laurence O. Whiteley
Pfizer Inc. Drug Safety Research and Development
Cambridge, Massachusetts
USA
Bonnie Wu
Biologics Development Sciences, Janssen Research and Development
LLC Pharmaceutical Companies of Johnson & Johnson Innovative Medicine
Spring House, Pennsylvania
USA
Jing Yuan
Department of Toxicology
Kymera Therapeutics
Watertown, Massachusetts
USA
Preface
Having dedicated more than a couple of decades to the development of biomarkers and bioanalysis in the realm of biologics, including monoclonal antibodies and recombinant protein therapies, we embarked on a career change with the anticipation that our wealth of experience could readily translate into the field of gene therapy drug development. However, what we hadn't fully grasped at the outset was the considerable complexity and formidable challenges associated with translational biomarkers, bioanalysis, and companion diagnostics when deploying adeno‐associated virus (AAV) as a vector to introduce transgenes, encompassing cDNAs and gene editing tools, into human subjects.
The successful advancement of a gene therapy drug necessitates the meticulous collection of pharmacokinetic and biomarker data to underpin efficacy and safety assessments, as well as the selection of suitable patients. The multifaceted nature of gene therapy, coupled with the vast troves of data involved, encompasses a wide spectrum of methods and technology platforms. This repertoire includes polymerase chain reaction (PCR)‐based techniques, such as quantitative PCR and digital PCR, for scrutinizing viral biodistribution and shedding patterns, reverse transcription‐PCR for analyzing transgene expression, enzyme activity assays, mass spectrometry, immunohistochemistry/in situ hybridization, and immunoassays for evaluating target engagement, substrate interactions, and distal pharmacodynamic biomarkers.
Moreover, ligation‐mediated (LM)‐PCR and linear amplification‐mediated (LAM)‐PCR are indispensable for the in‐depth analysis of recombinant AAV integration, while next‐generation sequencing (NGS) is employed to assess off‐target gene editing activity. The assessment of humoral antibody response and cellular immune response to AAV capsid and transgene products requires the application of anti‐drug antibody and neutralizing antibody assays, as well as ELISpot technology.
In addition, the evolving landscape of companion diagnostic development, particularly in relation to the anti‐AAV antibody screening assay supporting clinical studies and drug approval, presents unique and rapidly evolving challenges. Furthermore, as clinical data continues to emerge from ongoing trials, the regulatory environment governing the evaluation of efficacy and safety in the gene therapy field is in a state of flux.
Over the past decade, the discovery and development of AAV gene therapy medicines have gained remarkable momentum. This surge in growth, marked by a proliferation of preclinical studies and clinical trials, has led to a shortage of qualified researchers in translational sciences. In this dynamic landscape, the adoption of best practices in biomarker and bioanalysis, combined with up‐to‐date knowledge of regulatory guidelines, is of paramount importance. Such information is invaluable for gene therapy developers, whether they are working in academia, industry, or government organizations, as it equips them with the timely insights required to navigate the constantly evolving challenges and opportunities in this dynamic field.
January 2024
Yanmei LuSangamo Therapeutics
Boris GorovitsGorovits BioSolutions, LLC
Section IIntroduction
1Introduction to AAV‐based in vivo Gene Therapy
Oscar Segurado
ASC Therapeutics, Milpitas, CA, USA
1.1 Introduction
1.1.1 History of Gene Therapy
Watson and Crick first characterized the structure of DNA as a double helix in 1953 [1]. X‐ray crystallography of DNA, performed by Franklin, confirmed this finding [2]. Knowing DNA’s structure helped elucidate its functions, such as how it holds genetic information, can be copied, and gives rise to various proteins.
Although adeno‐associated viruses (AAVs) were discovered in the 1960s [3], they would not be used as genetic vectors until the 1980s. The first attempt at genetic manipulation in humans is believed to be the work of Terheggen et al. in the 1970s. German scientists used the Shope papillomavirus in three children whose bodies were unable to produce arginase. Without arginase, arginine accumulates in the body, causing neurological and muscular defects. The virus, known to produce arginase, was injected intravenously (IV) in hopes that the genetic information from the virus could enter human cells, resulting in arginase production. Unfortunately, IV injections of the virus did not help any of the three sisters that had this rare disorder, and the youngest, who was given a larger dose as an infant, suffered a brief allergic reaction without any positive response to the treatment [4].
In the 1980s, retroviral gene therapy was in development [5–7], and the first recombinant AAV vectors were created [8]. Synthetic insulin was the first genetically engineered drug, reaching the market in 1982 [9]. Zinc fingers were discovered in 1985, later providing a method of targeted gene therapy through zinc finger nucleases (ZFNs) [10]. The hepatitis B vaccine was the first recombinant vaccine available in 1986 [11], and the discussion of the human genome project began two years later [12]. Also in 1988, the first genetically modified crop was grown in US fields [13].
In 1990, research began in the United States, studying human gene therapy [14]. Dolly, the sheep, was cloned in 1996 [15]. By the year 2000, around 400 gene therapies had been tested in clinical trials [16]. The first gene therapy was approved in China in 2003, using a replication‐incompetent adenovirus vector for treating advanced head and neck cancer [17]. Modified lentiviral vectors began emerging in clinical trials around this time as well [18]. In 2007, human‐induced pluripotent stem cells (iPSCs) were first isolated, and this method is now quite common, using genetic reprogramming to compare patient‐derived cells to isogenic control cells [19]. The first gene therapy was approved in Europe in 2012 using an adenovirus [16]. In 2013, CRISPR/Cas9 was developed, where it was first used as a research tool [20]; it was not until 2018 that the first clinical trial in humans utilizing this technology completed their enrollment. Patients with refractory non‐small‐cell lung cancer were treated with CRISPR‐edited T cells [21]. This timeline can be viewed in Figure 1.1.
In 2020, over 400 gene and genetically modified cell therapies were in development, and today (2022), there are over 1000 in recruitment or active studies (clinicaltrials.gov). Gene therapies may replace inadequate and complex therapies in the near future. For some diseases, it may be able to reduce the amount and, eventually, the cost of treatments a person needs. Thus, it is likely to benefit those with poor quality of life due to an untreatable condition or an intense therapy regimen the most.
1.1.2 AAV‐based in vivo Gene Therapy: A Revolution in Medicine
Despite gene therapies being developed and tested in the United States since the 1990s, only 26 cell and gene therapies have been Federal Drug Administration (FDA)‐approved until February 2023, seven of which are cord blood treatments (Table 1.1). Of the other 19 therapies, 14 are ex vivo cell therapies and five are in vivo gene therapy treatments. Genetic diseases, those driven by mutations in the human genome, are ideal targets for treatments using gene therapy modalities. Gene therapy can address diseases driven by well‐defined genetic abnormalities where the biological function of the altered or missing gene is well understood. In many cases, these are rare diseases with unmet medical needs, often requiring complex medical regimens with limited options for effective treatments. However, in recent years, gene therapies have been investigated for the treatment of non‐monogenic diseases, for example, cancers and degenerative diseases of the visual and nervous systems.
Figure 1.1 Timeline of scientific advances in gene therapy research [1].
Table 1.1 FDA‐approved cellular and gene therapies.
Name
Indication
Type
Manufacturer
Abecma (idecabtagene vicleucel)
Adult relapse or refractory myeloma after ≥4 prior therapy lines, including immunomodulatory agent, proteasome inhibitor, anti‐CD38 monoclonal antibody
Ex vivo
; Lentivirus vector
Calgene Corporation, a Bristol‐Myers Squibb Company
Adstiladrin
Adult high‐risk Bacillus Celmette‐Guerin‐unresponsive non‐muscle invasive bladder cancer with carcinoma in situ
Adenovirus vector
Ferring Pharmaceuticals A/S
HPC, Cord Blood; Allocord; Clevecord; Hemacord; HPC, Cord Blood – MD Anderson; HPC, Cord Blood – LifeSouth; HPC, Cord Blood – Bloodworks
Hematopoietic and immunologic reconstitution with disorders affecting the hematopoietic system that are inherited, acquired, or from myeloablative treatment
Hematopoietic progenitor cells
University of CO Cord Blood Bank; SSM Cardinal Glennon Children's Medical Center; Cleveland Cord Blood Center; Duke University School of Medicine; NY Blood Center; MD Anderson Cord Blood Bank; LifeSouth Community Blood Centers; Bloodworks
Breyanzi
Adult large B‐cell lymphoma, including diffuse not otherwise specified high‐grade primary mediastinal and follicular grade 3B
Ex vivo
; Lentivirus vector‐modified autologous CD4+ and CD8+ T cells
Juno Therapeutics, Inc., a Bristol‐Myers Squibb Company
Carvykti (ciltacabtagene autoleucel)
Adult relapse or refractory multiple myeloma after ≥4 prior therapy lines, including proteasome inhibitor, immunomodulatory agent, anti‐CD38 monoclonal antibody
Ex vivo
; Lentivirus vector‐modified autologous T cells
Janssen Biotech, Inc.
Gintuit
Topical (non‐submerged) application to surgically created vascular wound bed in adult mucogingival conditions
Ex vivo
; Scaffold product of neonatal foreskin allogeneic fibroblasts & keratinocytes
Organogenesis, Inc.
Hemgenix
Adult hemophilia B (Factor IX deficiency)
AAV vector
CSL Behring LLC
Imlygic (talimogene laherparepvec)
Local treatment of unresectable, cutaneous, subcutaneous, and nodal lesions with melanoma recurrent after initial surgery
Modified HSV‐1 isolate with oncolytic activity toward tumor cells (JS1)
BioVex, Inc., a subsidiary of Amgen, Inc.
Kymriah (tisagenlecleucel)
Adult relapsed or refractory follicular lymphoma after ≥2 lines of therapy
Ex vivo
; Lentivirus vector‐modified autologous T cells
Novartis Pharmaceuticals Corporation
Laviv (Azficel‐T)
Improvement of adult moderate‐to‐severe nasolabial fold wrinkle appearance
Ex vivo
; Autologous fibroblasts
Fibrocell Technologies
Luxturna
Biallelic RPE65 mutation‐associated dystrophy
Recombinant AAV serotype 2 vector expressing RPE65
Spark Therapeutics, Inc.
Maci
Repair of adult single or multiple symptomatic, full‐thickness cartilage defects of the knee
Ex vivo
; Autologous knee cartilage chondrocytes in resorbable porcine type I/III collagen membrane
Vericel Corporation
Provenge (sipuleucel‐T)
Asymptomatic or minimally symptomatic metastatic castrate‐resistant (hormone refractory) prostate cancer
Ex vivo
; Autologous cellular immunotherapy
Dendreon Corporation
Rethymic
Immune reconstitution in pediatric congenital athymia
Ex vivo
; Allogeneic thymus from <9 months old heart surgery patients
Enzyvant Therapeutics GmbH
Skysona (elivaldogene autotemcel)
Slow progression of neurologic dysfunction in boys 4–17 years with early, active cerebral adrenoleukodystrophy
Ex vivo
; Lenti‐D lentivirus vector modified autologous CD34+ ‐enriched hematopoietic stem cells
Bluebird Bio, Inc.
Stratagraft
Adult thermal burns containing intact dermal elements, which surgical intervention is clinically indicated (deep partial‐thickness burns)
Ex vivo
; Allogeneic cultured keratinocytes and dermal fibroblasts in murine collagen type I scaffold
Stratatech Corporation
Tecartus (brexucabtagene autoleucel)
Adult relapsed or refractory mantle cell lymphoma and B‐cell precursor acute lymphoblastic leukemia
Ex vivo
; Gammaretrovirus vector modified antigen‐specific autologous T cells
Kite Pharma, Inc.
Yescarta (axicabtagene ciloleucel)
Adult B‐cell lymphoma refractory to first‐line chemoimmunotherapy or relapse within 12 mo. Of first‐line chemoimmunotherapy
Ex vivo
; gammaretrovirus vector modified autologous T cells
Kite Pharma, Inc.
Zynteglo (betibeglogene autotemcel)
Adult and pediatric beta‐thalassemia requiring red blood cell transfusions
Ex vivo
; BB305 lentivirus vector modified autologous CD34+ ‐enriched hematopoietic stem cells
Bluebird Bio, Inc.
Zolgensma (onasemnogene abeparvovec‐xioi)
Spinal Muscular Atrophy (Type I)
Recombinant AAV vector
Novartis Gene Therapies, Inc.
Information obtained on 1 February 2023 from https://clinicaltrials.gov/ct2/results?term=AAV+gene+therapy&Search=Apply&age_v=&gndr=&type=&rslt=.
Of the approved genetic therapies, five utilize lentivirus vectors, two use gammaretrovirus vectors, one uses an adenovirus vector, and three use an AAV vector. Two hundred and seven AAV gene therapy clinical trials can be found on the clinicaltrials.gov website, 111 of which are active trials, either in recruiting, enrolling, or collecting and analyzing data phases (Table 1.2). The majority of the AAV‐based gene therapy trials are documented as Phase 1 or 2 trials (some are combined phase trials, i.e. Phase I/II). Sixty AAV‐based gene therapy trials have been completed. Indications for completed AAV‐based gene therapy trials include muscular disorders, neurodegenerative diseases, retinal diseases and other visual defects, lysosomal and glycogen storage disorders, blood coagulation disorders, cardiovascular diseases, amino acid metabolism disorder, and arthritis (Table 1.3). The active trials include the above diseases and other indications such as infections, adrenal diseases, developmental disorders, hearing loss, and cancer. Some early success has been seen in neurodegenerative diseases, such as Parkinson’s and Alzheimer’s disease; however, this is likely limited by the number of remaining neurons, many of which are lost prior to even diagnosis [1, 2].
Table 1.2 AAV gene therapy clinical trial status.
# of trials
AAV Gene Therapy
207
Status
Not yet recruiting
13
Recruiting
57
Enrolling by Invitation
8
Active, not recruiting
46
Terminated
17
Completed
60
Unknown
6
Phase
Early Phase 1
3
Phase 1
138
Phase 2
109
Phase 3
20
N/A
7
Information obtained on 1st February 2023 from https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/approved-cellular-and-gene-therapy-products.
Table 1.3 Complete and active AAV gene therapy clinical trial indications.
Indications of clinical trials
Complete
Active
Muscular Disorders
12
8
Vision Defects
10
4
Neurodegenerative Diseases
10
13
Retinal Diseases
9
25
Lysosomal & Glycogen Storage Disorders
7
20
Blood Coagulation Disorders
5
20
Cardiovascular Diseases
2
4
Amino Acid Metabolism Disorders
1
5
Arthritis
1
1
Infections
3
Adrenal Diseases
3
Developmental Disorders
2
Digestive System Diseases
3
1
Hearing Loss
1
Cancer
1
Despite its promise and setbacks, gene therapy’s potential across therapeutic areas remains enormous, offering the hope of “one and done” cures for serious diseases with significant unmet needs. AAV‐based gene therapy has received the most attention in basic and clinical research recently, moving rapidly into the biotechnology industry, resulting in clinical trials designed to prove its efficiency, safety, cost‐effectiveness, and range of use. Recombinant AAVs (rAAVs), engineered capsids with lower immunogenicity, the addition of synthetic promoters, and gene editing techniques are just some of the developments in AAV vector‐based gene therapy. Although these developments have come to fruition relatively quickly, many challenges remain when developing and using gene therapy treatments. Several gene editing techniques have emerged over the past two decades, which have been crucial in researching and developing disease mechanisms, therapies, and more. Although there have been setbacks, the promise of gene therapies and the potential of direct gene editing remains. The technological advancements in labs, clinics, and manufacturing processes continue to make it more and more possible for long‐lasting rare disease treatments. Thus, it is crucial to understand how AAV‐based gene therapies are created and implemented, the benefits and risks of these treatments, as well as the identification of their most relevant indications.
1.1.3 The AAV Vector Structure
The AAV is part of the Parvoviridae family, being approximately 25 nm in diameter with its DNA contained within an icosahedral capsid, the protein shell. Single‐stranded DNA of approximately 4.7 kb is composed of genes for three capsid proteins [22] four rep proteins, and an assembly‐activating protein [23]. Palindromic inverted terminal repeats (ITRs) are located at each end [24], forming T‐shaped hairpin structures. The Rep gene is required for DNA replication and packaging, and the Cap gene encodes the capsid assembly proteins. In studies of AAV2, it was found that the structural proteins, VP1, 2, and 3, exist in a 1 : 1 : 10 ratio, forming an icosahedral, symmetrical shape [25]. VP1 was essential for infection while VP2 was necessary for nuclear transfer of capsid proteins [26]. VP3 was found to allow the binding of the virus to cell receptors [27, 28].
The AAV was discovered in 1965 as a contaminant of an adenovirus preparation from rhesus monkey kidney cell cultures infected with simian adenovirus type 1 [3]. AAVs rely on helper viruses in order to replicate in mammalian cells, typically adenoviruses or herpes viruses.
When using AAV as a vector, the DNA of interest replaces the genomic DNA, the Rep and Cap genes, between the ITRs [29]. The exogenous DNA is referred to as the transgene expression cassette. When creating a recombinant AAV, the gene expression cassette needs to include a promoter, transgene, and a termination signal, which is specific to the goals of that particular gene therapy. Figure 1.2 displays the requirements for creating a rAAV vector. Tissue and cell‐type specificity, packaging size limits, and necessary expression level all need careful consideration when creating the gene expression cassette.
Figure 1.2 Creation of recombinant AAV particles.
However, the baculovirus‐infected insect (Sf9) system is increasingly used with scale‐up production. Specifically, the OneBac system has been developed which integrates rep and cap into one baculovirus, resulting in a decrease in the required number of baculovirus particles for efficient rAAV production [30].
1.1.4 Cell Entry and Transduction Pathway
Wild‐type AAV viruses can infect dividing and nondividing cells. The majority of the AAV genomes, wild‐type and recombinant, exist as circular episomes in tissues, though integration into the host cell chromosomes at AAV integration sites can happen in low frequency [31]. AAV will not replicate without Rep proteins, which regulate viral transcription. Rep proteins can be provided by a helper virus and remain latent until this occurs [32]. Once the helper virus is delivered, the Rep genes are expressed and replication can occur via the rolling hairpin mechanism, where a Rep protein binds the Rep binding element (RBE) within the hairpin, followed by duplication of the DNA strand and packaging. The cell entry and transduction process of an rAAV is shown in Figure 1.3. Cellular transduction of an AAV vector begins with viral capsid interaction with the target cell’s receptors. The cell uses endocytosis (clathrin‐coated pits) to internalize the virus, which then enters the endocytic/proteasomal compartment. These may be critical steps for transduction because injection of AAV2 directly into cells resulted in a reduced infection rate compared to exposure [33]. Interestingly, the efficiency of transduction is largely affected by acidity of the endosomal compartment [34]. Proteasome inhibitors also increase transduction efficiency [35].
Figure 1.3 rAAV vector entry and transduction pathway. (1) AAV vectors bind to receptors of target cells, (2) initiating endocytosis via clathrin‐coated pits. (3) The endosomal compartment contains the AAV, (5) which can escape to the nucleus through the nuclear pore. (6) Uncoating releases single‐stranded DNA (7) for a second strand to be synthesized (de novo synthesis or base pair strand annealing). (8 and 9) This leads to the production of the corrected/therapeutic mRNA and subsequent protein to be produced.
The virus escapes the endosome and is imported through the nuclear pore complex into the nucleus, undergoes capsid uncoating, and the DNA is converted into double‐stranded DNA via de novo synthesis or base pairing. The DNA can then be transcribed and expressed [36]. Particles that are not able to translocate to the nucleus are degraded by the proteasome and presented to cytotoxic T cells [37]. Thus, prior to genome integration, intracellular trafficking involves a number of events in which breakdown at any step can result in failure of gene delivery.
De novo synthesis of the complementary DNA strand and strand annealing result in the conversion of single‐stranded DNA to double‐stranded DNA. Strand annealing involves the base pairing of a coinfected separate AAV complementary single‐strand genome. Due to the synthesis of the second DNA strand being considered the rate‐limiting factor in transduction efficiency, second‐generation AAV vectors with double‐stranded DNA have been developed, called self‐complementary AAV vectors, which have been shown to be safe and reliable for organ‐specific transduction [38, 39].
1.2 Advantages and Disadvantages for AAV in vivo
1.2.1 Effectiveness and Advantages of AAV Vectors for in vivo Gene Therapy
AAV vector has been labeled as the safest and most effective vehicle for the delivery of in vivo gene therapies that produce long‐term expression with a single injection. rAAV effectiveness is largely determined by the interaction of the capsid and the cell surface receptors as well as downstream events after internalization. Transduction efficiency can be altered due to the endosomal compartment pH and the use of proteasome inhibitors [34, 35]. Immune reactions to AAVs significantly impact a gene therapy’s efficiency and will be discussed further in Section 1.5.3. Addressing Immunogenicity and Chapter 5.
There are many advantages to using AAV vectors for gene therapy. The human native AAV is not known to cause disease, has no pathogenicity [3], and has the ability to infect numerous mammalian cell types. AAVs efficiently deliver genetic material with low toxicity and immunogenicity, a good safety profile, and long‐term effects [40, 41].
1.2.2 Challenges of AAV Vectors for in vivo Gene Therapy
Challenges, of course, still exist for gene therapy using AAV vectors. Large‐scale manufacturing, particularly quality control and standardization, is difficult to maintain for gene therapies, resulting in high costs. Although AAVs provide efficient gene delivery, they are less immunogenic than adenoviruses. Before transduction, single‐stranded DNA from AAVs must first be made into double‐stranded DNA. AAVs have limited capacity, holding approximately 4.7 kilobases (kb) [22]. Implementing a split vector approach can bypass the limitation of small capacity; however, serogenicity and type of cell or tissue can markedly affect the expression of split vectors. Also, replacing Rep genes with exogenous DNA lowers the transduction frequency because, without them, ITRs have little enhancer and promotor activity. Self‐complementary vector AAVs can be implemented to improve efficiency and onset of gene therapies.
There are potential sources of toxicity that must be considered in the development of AAV gene therapies, insertion mutagenesis, tumor induction, and immune response, dependent upon reactions to the capsid and DNA of the AAV. Toxicity has been reported for AAV clinical trials, causing hepatotoxicity, muscle toxicity, thrombotic microangiopathy, etc. [42]. Route of administration and number of viral particles used for gene therapy may contribute to toxicity and the immune response as well. In two studies testing a hemophilia B gene therapy, AAV gene transfer in the liver resulted in the development of capsid‐specific CD8+ T cells and elimination of transduced hepatocytes. The reaction was halted by oral corticosteroids [40, 43], although in some cases, immune‐modulating agents have been shown not able to prevent loss of transgene expression. Reactions to natural AAV capsids may predict the reaction and effectiveness of AAV‐based gene therapies [44]. Transient B‐cell depletion and inducing immune tolerance with rapamycin may be good strategies to reduce the host immune response to AAVs, though better strategies may be necessary to prevent a decrease or, in some cases, a complete loss in transgene expression.
Immunogenicity and malignancy are other challenges that must be addressed during development and preclinical studies to ensure patient safety. Manufacturing gene therapies can be challenging because of the quality control needed throughout and production costs. Scalability has been a major obstacle to gene therapy production. Due to the time it takes for clinical trials and regulatory procedures, drugs take ample time to reach the market, even for diseases without any current treatment options.
1.3 Technology Platforms of AAV‐based in vivo Gene Therapy
Genetic editing is the act of revising, removing, or replacing DNA directly, which can be used to tailor recombinant AAVs for specific gene therapies. Gene therapy uses gene replacement, gene addition, genome editing, and gene regulation techniques. Typically, ZFN, transcription activator‐like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) are used for various gene editing techniques. Each technology has specific uses and limitations, which are discussed in brief in this chapter. Further detail will be provided in Chapter 2.
1.3.1 cDNA Replacement
cDNA replacement is the method used in gene replacement therapies to express a functional copy of a defective gene episomally, directed by an exogenous promoter, which results in stable gene expression. Most programs use AAV to deliver vectors in gene replacement, although this method is limited to recessive or haploinsufficient disease targets. Artificial promoters allow for specificity but can compromise physiological gene expression.
1.3.2 Genome Editing
Breaks and DNA repair are used in genome editing to design specific, targeted vectors. Genome editing can be performed using various platforms, including ZFN, TALEN, CRISPR/Cas9, base and primer editing, and RNAi gene silencing. These technologies will be introduced next in this chapter with more detail explained in Chapter 2.
Many gene editing techniques utilize a DNA nuclease to cleave a specific site in the genome (Figure 1.4). ZFNs and TALENs have been used previously but are challenging and time‐consuming to design or have large constructs with limited transduction. Genome editing platforms can be combined with viral vectors to improve a gene therapy’s efficacy and lower the risk of off‐target effects.
Figure 1.4 Gene editing techniques using double‐stranded breaks. ZFN and TALEN use DNA recognition protein motifs and fused with a cleaving enzyme (FOK1). CRISPR/Cas9 uses a guide RNA that binds to a target sequence, then Cas9 binds the guide RNA to cleave both DNA strands.
1.3.2.1 ZFN
Targeted gene replacement was first developed using ZFNs. Both ZFN and TALEN technologies pair protein domains with nucleotides with a cleaving enzyme to create a double‐stranded break (DSB). Approximately 30 amino acids make up one zinc‐finger motif, each able to bind three nucleotides. ZFNs generally display lower specificity than TALENs [45, 46] and relatively similar specificity to improved CRISPR/Cas9 methods with certain guide RNAs (gRNAs) [47–49]. However, specificity can be difficult to compare and depends on the specific technological methods, including the gene editing target and nuclease architecture. It also produces less immune reaction compared to the newer CRISPR technology. In human studies using ZFN for gene editing was found to have a good safety profile with evidence of successful gene editing, although sustained expression was not detected [50].
1.3.2.2 TALENs
TALENs make use of non‐specific DNA nucleases bound to a DNA‐binding domain that can target a specific sequence. TALENs were more quick and efficient to use compared to earlier technologies [51]. The first use of TALEN technology in human treatment was in 2015, treating pediatric acute B lymphoblastic leukemia with TALEN‐engineered CAR T cells [52]. TALEN can be advantageous for difficult‐to‐edit regions of DNA where it can be more efficient than CRISPR [53]. One disadvantage of the TALEN system is its incompatibility with AAV vectors. In general, the targeted use of TALEN in vivo is challenging because of its large size and repetitive characteristics. Using high‐capacity adenovirus or non‐viral delivery is best when implementing the TALEN system directly in vivo[54].
1.3.2.3 CRISPR/Cas9
CRISPR/Cas9 is a robust gene editing tool that has emerged as the preferred gene‐editing technique because of its ease of use, low cost, and high efficacy. While ZFN and TALEN are protein‐based DNA recognition techniques, CRISPR is an RNA‐based technique. In brief, CRISPR/Cas9 binds to DNA fragments containing protospacer‐adjacent motif (PAM) sites and creates DSBs in the DNA. A gRNA locates the gene of interest or location within the DNA and a Cas9 nuclease induces a DSB in the DNA. Then, DNA repair via non‐homologous end joining (NHEJ) or homologous direct repair (HDR) is initiated [55].
As with most gene editing techniques, problems arise, particularly off‐target effects and immunity. Off‐target effects are an inherent issue in gene editing, being identified with use of ZFNs, TALENs, and CRISPR/Cas9 [56, 57]. Cas9 from bacteria, used on the human genome significantly increases the chance of off‐target effects because the genome is much larger than that of bacteria [47]. Thus, many improvements in the CRISPR/Cas9 system have aimed at reducing off‐target effects and providing stable genome expression. In 2007, cells treated with CRISPR were found to activate adaptive immunity, inspiring many improvements and modifications to the system [58].
Improving gRNA is another important area of gene therapy development because it has significant effects on sensitivity and specificity of the system. Truncated gRNA, or gRNA lengthened by two guanine nucleotides, has been found to reduce off‐target effects [48, 49, 59]. Many tools have been developed to design gRNA and detect off‐target effects in vivo[60]. Integrase defective lentiviral vectors‐capture was one of the first assays created to monitor off‐target effects in vivo following the use of ZFNs, and GUIDE‐seq is the common tool used to identify off‐target sites with CRISPR/Cas9 treatments [61–63]. The use of nickases to create a single‐stranded rather than a double‐strand break also greatly reduces off‐target effects [49].
1.3.3 Base Editing and Prime Editing
As mentioned, base and prime editing do not necessitate any DSB, making it safer and more accurate than classical gene editing technologies. Base editing is a tool for permanently correcting base pair mismatches [64]. A more recently developed method called prime editing uses prime editing gRNA (pegRNA) and Cas‐nickase, which can correct transition mutations, unlike base editing [65].
Base editors are classified as cytosine or adenine base editors, which need an inactive Cas or Cas nickase coupled with deaminase to make the edit and a gRNA to guide Cas to the targeted DNA binding site. Dual base‐editor systems have also been created for a combinatorial editing approach [66]. Creating precise base edits of more than four transition mutations has not been feasible with base editing, leading the path to prime editing. An engineered reverse transcriptase bound to a Cas9 nickase and a prime editing gRNA are necessary for prime editing [65]. PegRNA not only holds the complementary sequence but also another sequence directing the exact sequence change. These methods will be very useful in editing large genes and the treatment of autosomal dominant diseases [67]. Because these technologies are quite new, much research is needed to determine their full safety and efficacy in a variety of uses.
1.3.4 RNAi Gene Silencing
Gene silencing can be performed with RNA interference (RNAi). RNAi can be utilized for gene regulation through the knockdown of a target gene, known as gene silencing. This occurs through a process where double‐stranded RNA (dsRNA) is processed into short interfering RNAs (siRNAs), resulting in transcriptional or post‐transcriptional gene silencing by degradation of complementary mRNA [68]. dsRNA of more than 30 base pairs can result in a significant antiviral response and apoptosis [69]. Probably the most promising area for RNAi is in antiviral strategy for infections [70].
1.3.5 Gene Addition
Gene therapy for more complex genetic diseases, infectious diseases, and cancer can be performed via gene addition methods to over‐express an endogenous or synthetic gene. A herpes simplex virus (HSV) vector has been developed containing the granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) gene to help immune effector cells attack tumor cells [71]. Another example of gene addition has been done by transferring a CAR gene into T cells ex vivo for treating B‐cell malignancies [72].
1.4 AAV Serotypes and Tissue Affinity
Twelve AAV serotypes have been identified, and over 100 variants have been found in human or nonhuman primate tissues; although, the best characterized is AAV2 [29]. Optimal serotypes are specific to tissue type, including AAVs 1, 2, 4, 5, 8, and 9 for the central nervous system (CNS), AAVs 1, 8, and 9 for the heart, AAV2 for the kidney, AAVs 7–9 for liver, AAVs 4–6 and 9 for the lung, AAV8 for the pancreas, AAVs 2, 5, and 8 for photoreceptor cells, AAVs 1, 2, 4, 5, and 8 for retinal pigment epithelium, and AAV1 and 6–9 for skeletal muscle. The varying serotypes with differing tissue specificity and infection rates, make AAV vectors a great candidate for gene therapy. AAV gene therapies commonly target the liver, CNS, and muscle, which can become biofactories for producing the required protein.
Other characteristics specific to an individual or a few AAV serotypes include their transduction efficiency, homology to other serotypes, and speed of tissue targeting. AAV1 and 6 share 99% homology, while AAV2 has close homology to most serotypes, not including AAV4, 5, 11, and 12. AAV 8 and 10 share 93% homology, and AAV11 and 12 have close homology to AAV4. AAV5 shares the least homology with other serotypes. Zincarelli et al. found that AAV2‐5 have low transduction efficiency, and AAV3 and 4 are slow in targeting tissues [73], but this varies significantly depending on cell type [74].
1.4.1 The Liver as a Biofactory
The liver is targeted in gene therapies of hemophilia for the production of factor VIII and factor IX, missing coagulation proteins [75]. Hemophilia is an ideal target for gene therapy, especially as the genetics and pathophysiology of the disease are well understood. The goal of gene therapy is the long‐term expression of the missing genes in the coagulation cascade at levels high enough to be therapeutic, a so‐called functional cure. A growing body of clinical experience supports the use of liver‐directed rAAV as a gene therapy transporter of the replacement gene for the treatment of hemophilia [75, 76].
1.4.2 The CNS as a Biofactory
Some neurodegenerative disease gene therapies have been successfully developed for spinal muscular atrophy (SMA) and are being developed for other CNS diseases, including Parkinson’s and Alzheimer’s disease and ALS. The blood–brain barrier (BBB) tends to be an obstacle for AAVs. Two AAVs are known to cross the BBB; IV injection of AAV9 and AAVrh.10 was shown to cross the BBB more effectively than AAV2 or AAV8 to transduce neurons and glial cells [77]. More research is critical to determine how the CNS can be targeted efficiently to use as a gene therapy biofactory for CNS diseases and disorders.
1.4.3 The Muscle as a Biofactory
Muscular dystrophies are another area of interest for gene therapy. AAV8 and AAV9 tend to be used for targeting muscle, although AAV1, 2, 5, 6, and 7 can also be utilized [78]. Muscle can be used as a biofactory, producing secretory factors for infectious diseases, diabetes, atherosclerosis, hemophilia, and cancer therapeutic agents. Muscle cells display the ability to secrete recombinant therapeutic proteins into the bloodstream after intramuscular AAV transduction, making it a useful tool for the treatment of distant organs [17].
1.5 Precision Medicine: Screening and Monitoring Biomarkers, Companion Diagnostics
Besides standard efficacy and safety assessments using clinical outcomes, usually primary endpoints in clinical trials, the incorporation of biomarkers can support the monitoring of patients on gene therapies. These biomarkers include translational, structural, functional, and tumorigenesis biomarkers, which can be used in diagnostics, safety, response, and monitoring. A more detailed discussion can be found in Chapter 3.
Translational biomarkers support the translation from data obtained in preclinical research to clinical trials. These assessments address the sensitivity, specificity, and toxicity of the AAV‐based gene therapy and inform measures of therapeutic efficacy and prognosis. Structural biomarkers assess tissues and organs through imaging methodologies, such as ultrasound, MRI, and PET. Functional biomarkers can assess clinical endpoints related to organ functionality and prognosis, such as motor and cognitive clinical assessments in various CNSCNS and muscular degenerative diseases. Liver and kidney function biomarkers can be assessed in the blood to measure their function and monitor treatment responses of liver and kidney‐targeted gene therapies. Preexisting antibodies to AAV can be used as biomarkers for the prediction of AAV gene therapy effectiveness and often need to be developed into companion diagnostics. AAV antibodies present may be a predictor for AAV gene therapy response and can indicate a need for adjuncts to prevent the rejection of transfected cells. Tumorigenesis biomarkers can assess the potential development of neoplasia. Tumor testing and profiling biomarkers can help identify the type of tumor and oncogenesis. Genetic testing can determine a person’s risk of certain cancers or the type of cancer‐causing tumor. Genetic testing, specifically of a tumor, can give further information about its growth and other characteristics.
1.5.1 Gene Therapy Clinical Trials: Spotlight on Hemophilia A
As a representative example of the clinical development processes for gene therapies, we will reference the trial design for patients with severe or moderately severe forms of hemophilia A. This has been extensively reviewed by Pipe et al., showing that, in contrast to several biopharmaceutical companies developing gene replacement therapies based on the B‐domain deleted human factor VIII, ASC therapeutics is introducing a second‐generation gene therapy based on a B‐domain deleted human/porcine chimeric factor VIII [79]. This next‐generation gene therapy has shown in preclinical studies the potential to decrease dose requirements, increase durability through improved factor VIII expression, and improve durability driven by a more efficient expression and reduced intracellular stress.
A first‐in‐human, open‐label, dose‐finding study is designed to assess the safety and preliminary efficacy of a single infusion of the AAV8‐based chimeric transgene replacing the coagulation protein factor VIII in study participants with severe and moderately severe hemophilia A (FVIII activity ≤ 2 International Units/Deciliter).
In this study, the main safety monitoring parameters include:
1)
Physical examination, including assessments of general appearance; head, eyes, ears, nose, and throat; the cardiovascular, dermatologic, lymphatic, respiratory, gastrointestinal, genitourinary, musculoskeletal, and neurologic systems. Height and weight will also be measured and recorded.
2)
Vital signs, including oral temperature, pulse rate, respiratory rate, and blood pressure.
3)
Viral shedding, assessing the evidence of potential viral transmission, will be tested on samples of blood, saliva, urine, stool, and semen.
4)
Liver ultrasound.
The main efficacy monitoring assessments include:
1)
FVIII activity, determined by validated assays, one‐stage
activated partial thromboplastin time
(
aPTT
), and chromogenic FXa. FVIII levels should be taken at a trough or close to trough levels, meaning after a minimum of 72 hours has elapsed since the last infusion of FVIII protein concentrates.
2)
Bleeding episodes and FVIII replacement therapy will be captured in the participant’s diary. In addition, the number of bleeding episodes requiring treatment, and the number of bleedings that do not require treatment following the administration of ASC618 infusion, will be recorded throughout the study.
The main exploratory assessments include:
1)
Hemophilia A quality of life
(
Haem‐A‐QoL
) questionnaire was completed by participants during the study.
2)
