Points to Consider for Human Gene Therapy and Product Quality
Control State Food and Drug Administration of China
This document by Shenzhen SiBiono GeneTech Co., Ltd. and the National Institute for the Control of Pharmaceutical and Biological Products, China, is now SFDA’s official guidance regarding gene therapy development. It was translated from the Chinese by Shenzhen SiBiono GeneTech Co., Ltd. (Also see the interview with SiBiono founder, Dr. Zhaohui Peng, page 42).
May 1, 2004
By: Zhaohui Peng, State Food and Drug Administration of China
BioPharm International
Introduction
Gene therapy is a medical intervention based on the modification of the genetic material of living cells. Currently, gene therapy is restricted in application to somatic cells.
Based on transferring methods, gene therapy can be classified into two categories: ex vivo and in vivo. ex vivo gene therapy refers to cells being modified ex vivo for subsequent administration to humans, while in vivo refers to cells being altered in vivo by giving gene therapy directly to the subject. The products of ex vivo gene therapy are cells that are modified and are intended to be administered to the patient. ex vivo gene therapy is expected to be performed in well-established medical care establish- ments with specially trained medical professionals and GMP facilities. The products of in vivo gene therapy are recombinant DNA or RNA in the form of naked DNA, DNA complex, or viral vectors that are manipulated by genetic technologies. Both ex vivo and in vivo gene therapy products are subject to the regulations in this guidance. Because of the complexities of the different modalities of gene therapy, it is not possible to generalize a common guidance that is suitable for all kinds of products. However, the following basic principles should be followed when sponsoring the development of a gene therapy product:
1 The safety and efficacy of the product should be guaranteed. A comprehensive assessment of the benefit and risk of the product should be conducted.
2 New and innovative ideas should be promoted when sponsoring gene therapy product development. Considering the uniqueness of gene therapy relative to traditional chemically synthesized and genetically engineered protein medicines, there will be certain flexibilities for the regulation of novel gene therapy products. Gene therapy as a form of medical intervention is still in its early phase of development. SFDA expects the sponsors to not just follow this guidance but also to conduct rigorous scientific study to ensure the development of a safe and efficacious gene therapy product.
When applying to conduct a clinical trial of an investigational new gene therapy drug (IND) from the State Food and Drug Administration (SFDA), the sponsor should prepare the application following the guidance in this document. In addition, the following information should be provided:
1 Review of the research and development of the relevant gene therapy field
The review should cover the following topics:
* therapeutic gene
* delivery vehicle
* gene delivery system and method
* in vivo study efficacy data
* preclinical animal study, including toxicity, safety, and efficacy data
* clinical trial investigation plan, including safety and efficacy study
* overview of production process
* overview of quality control
* discussion of the novelty of the product
* discussion of the product commer-cialization strategy.
The review should be comprehensive and up to date.
2 Review of the intellectual property status of the product
The review should cover the following topics:
2.1 Overview of the intellectual property status of the proposed product
2.2 Reference and patent search report
The reference and patent search should cover the therapeutic gene, delivery vehicle, delivery product, and other components in the final product, producer cell and production process, and related topics.
Application Content and Product Quality Control
The following should be included in the application proposal:
1. Construction of the DNA expression cassette and the gene delivery system
1.1 Therapeutic target gene
A detailed description of the clonal origin of the therapeutic target gene, including a patent search on the gene, should be provided. Method of gene cloning and sequence identity should be provided.
1.2 Vector
Information supplied should include restriction mapping and gene-bank data for the vector. Known regulatory elements such as promoters, enhancers, and PolyA should be identified. If there is any change in the vector backbone gene structure (such as deletion, mutation, or insertion), the DNA sequence data should be provided. For a new viral vector, it is necessary to provide information on the material, method of construction, and testing of the new vector.
For non-viral gene delivery systems, plasmid is needed to express the target gene in human cells. In addition to naked DNA, another component is generally used to complex with the DNA. This guidance does not cover oligoribo-nucleotide (such as antisense RNA, ribozyme, and siRNA) products.
1.3 DNA expression cassette
A detailed description of the cloning procedure, the methods and materials used, and DNA sequence data should be given. Known regulatory elements such as promoters, enhancers, and PolyA should be identified. Restriction mapping of the gene expression cassette and the kinetics of gene expression should be provided.
1.4 Construction of the gene delivery system (including viral and non-viral gene delivery systems)
1.4.1 Viral gene delivery system including adenoviral vector, retroviral vector, and adeno-associated viral (AAV) vector
A thorough description of the clonal origin of the viral vector should be provided. The methods and materials used for the construction of the viral vector should be included. Testing methods and results should also be provided. General testing should include structural analysis (for example, restriction mapping and PCR), complete sequencing of the viral genome (≤ 40kb), gene expression and bioactivity analysis, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), DNA sequencing of the gene expression cassette, Western blot analysis of the expressed protein, transduction efficiency analysis, negative-staining transmission electron-microscopy of the purified viral vector, replication-competent virus detection, and analysis for residual process contaminants.
1.4.2 Non-viral gene delivery system
Non-viral gene delivery systems encompass naked DNA, mammalian cell carrier systems, and other carrier systems such as liposome, polypeptide, and gold particles. The nature and characteristics of the non-viral delivery system should be described adequately. To prevent allergic reaction to penicillin in some patient populations, it is recommended that a kanamycin- or neomycin-resistant gene be used as the drug selection gene.
For the physical delivery system, a detailed description of the delivery method, procedure, efficiency of gene delivery and expression, gene stability after delivery, and bioactivity should be included. Evidence of absence of gene arrangement and mutation should be provided. Detailed testing results should be given, including plasmid restriction mapping, PCR analysis, DNA sequencing of the gene expression cassette, SDS-PAGE, and Western blot analysis of the expressed protein.
2. Generation and characterization of cell banks and engineered bacteria banks
A three-tiered bank system should be established. These typically include a primary seed bank, a master cell bank, and a working cell bank. For engineered bacteria, these typically include a primary bacteria bank, a master bacteria bank, and a working bacteria bank.
2.1 Cell bank
2.1.1 Primary seed bank: The origin, cell passage history, and cell characterization, as well as cell culture and banking procedures should be described.
2.1.2 Master cell bank: The master cell bank should be derived from one or more ampoules of the primary seed bank by serial subculture to a specific passage number. The passage number should be identified.
2.1.3 Working cell bank: The working cell bank is derived from one or more ampoules of the master cell bank by serial subculture to a specific passage number. The passage number should be identified.
2.1.4 Characterization of cell banks: The master cell bank or working cell bank should be characterized according to guidelines in “Guidance for Application of Clinical Trials for Human Gene Therapy.”1
Additional testing should include:
a) testing of susceptibility of virus infection and production
b) testing the status and stability of the transduced gene and level of gene expression
c) testing for generation of replication-competent viruses.
If a helper virus is needed in the testing, the origin, methods of preparation and separation, and virus passage history should be provided.
2.2 Bacteria bank
A master bacteria bank and a working bacteria bank should be established and tested according to the specifications of the “Chinese Pharmacopoeia.”2
2.2.1 Testing of the master bacteria bank
a) Uniformity and the identity of bacteria host strain: Testing should include the origin, genotype and phenotype of the bacteria strain. The genotype should be tested by RAPD (random amplified polymorphic DNA). The phenotype should be tested for the specific marker or antibiotic resistant gene marker of the engineered plasmids.
b) Purity: The bacteria should be shown to be free of other adventitious agents, such as fungi and bacteria.
c) Stability: The ratio of transformed bacteria, propagation condition, copy numbers, level of gene expression, and passage number should be qualified.
d) Sequencing of gene expression cassette: The inserted gene of interest and associated regulatory elements should be sequenced.
2.2.2 Working bacteria bank
Testing of the working bacteria bank should be performed in accordance with the requirements for the master bacteria bank mentioned above with the exception of the sequencing of the gene expression cassette.
3. Manufacture of gene therapy products
3.1 General requirements
3.1.1 Description of manufacturing facility and environment
GMP regulations should be followed in the manufacturing of gene therapy products. This applies to both ex vivo cell products and recombinant viral products. Recombinant viral products intended for preclinical and clinical studies should be manufactured using a production process that can be validated.
3.1.2 A detailed description of the manufacturing process, raw materials, and the components used should be provided.
3.1.3 Manufacturing process controls should be provided. Critical process parameters should be identified, monitored, and recorded.
3.1.4 Batch production records and testing records of one lot of product should be provided.
3.1.5 One lot of product should be tested by institutes that are designated by SFDA for lot-release testing. The testing report should be provided.
3.2 Recombinant viral vector as gene therapy product
A master virus bank (MVB) and a working virus bank (WVB) should be established and tested. The WVB should be derived from the MVB. A detailed description of the origin, construction, cloning, passage, and storage of the virus bank should be provided. Quality control testing procedures outlined in this document should be followed. Production should be initiated from producer cells derived from the working cell bank. Non-qualified cells should not be used for production purposes. For a viral vector product, the producer cell infection should be carried using a virus from the WVB. Infection should not be performed using a non-qualified virus. Production can be performed using large-scale attachment- dependent or suspension cell culture. Virus banks can be prepared directly from a cell culture without further purification. Buffer formulation for virus banks should be qualified to protect virus infectivity during storage.
3.3 Non-viral plasmid DNA complex as gene therapy product
A detailed description of the following aspects should be provided:
3.3.1 Manufacturing process and quality control of the recombinant plasmid DNA
3.3.2 The origin, characteristics, and method of preparation of the liposome
3.3.3 The origin, characteristics, and method of preparation of the polypeptide
3.3.4 The origin, characteristics, and method of preparation of other components in the product complex
3.3.5 Method of preparation and quality control of the final product complex
For gene therapy products in the form of gene gun and other physical gene delivery approaches, detailed description of the following items should be provided:
a) origin and preparation of therapeutic gene
b) gene expression
c) propagation of the naked DNA
d) DNA purification
e) equipment characteristics
f) method of gene delivery.
3.4 Genetically-modified somatic cells as products
This category encompasses ex vivo gene therapy products. Manufacture of this category of products should follow guidelines in “Guidelines for Study of Somatic Cell Therapy and Quality Control.”3 Detailed description of the process for cell propagation, media used for cell culture, method of cell collection, method of ex vivo cell transduction and selection, and formulation used for washing and storage of the transduced cells should be provided.
4. Quality control
Extensive quality control testing should be performed during the manufacture of gene therapy products. Product release testing should be performed on the final drug product. However, depending on the nature of the manufacturing process, some testing should be performed on process samples if the excipients included in the final product formulation interfere with the testing.
4.1 Recombinant viral vector as gene therapy product
In reference to the domestic and international viral vector development status, testing of a recombinant adenovirus (rAd) product is used as an example of the quality control testing for viral vector products. The testing can be used as a reference for other viral vector products.
4.1.1 Quality control for rAd gene therapy product
4.1.1.1 Crude harvest
a) sterility tests to be performed in accordance with “SFDA Guidance for Biological Products, subpart
