CTNBIO NORMATIVE INSTRUCTION Nº 9, OF OCTOBER 10, 1997 Provides for the rules for genetic intervention in human beings The NATIONAL BIOSAFETY TECHNICAL COMMISSION - CTNBio, in the exercise of its legal and regulatory incumbencies, resolves: Article 1 The Genetic Intervention in Human Beings shall follow the rules set forth in this Normative Resolution. Article 2 This Normative Resolution comes into force on the date of its publication. LUIZ ANTÔNIO BARRETO DE CASTRO Publication by the Federal Official Gazette (D.O.U.) of October 16, 1997, Section I, pages 23.487/23.488. EXHIBIT RULES ON GENETIC INTERVENTION IN HUMAN BEINGS 1. Preamble A. Every trial of genetic intervention or manipulation in human beings shall be deemed Research with Human Beings and, as such, qualified under Resolution No. 196/96 of the National Health Council, complying with the principles of autonomy, non-maleficence, beneficence and justice. Solely proposals that fulfill all requirements of the aforementioned Resolution No. 196/96 shall be analyzed, as detailed below. B. Solely proposals of genetic intervention or manipulation in human beings involving somatic cells shall be analyzed. Any genetic intervention or manipulation in human germination cells is forbidden, as provided for by article 8 of Law No. 8.974 of January 05, 1995 and Normative Resolution No. 8/97 of CTNBIO. C. All proposals of genetic intervention or manipulation of human beings shall be analyzed by CTNBio from the viewpoint of two major biosafety risks, to wit: (1) risk of horizontal transmission of the transferred nucleotide sequence or of the vector to other persons with which the patient has contact, and (2) risk of unintentional modification of germination cells, with vertical transmission of the genetic alterations to the patient’s progeny. 2. Scope As provided for by article 8 of Law No. 8.974/95, any intervention in in vivo genetic material is forbidden, except for treatment of genetic defects. Genetic defects mean those inherited or acquired during life that may cause problems to the human health. Genetic defects may be caused by: point mutation, chromosomal insertion, deletion, translocation, amplification, loss or gain, or by the presence of the genome or a part of the genome of infectious organisms. Somatic gene therapy or gene transfer to somatic cells are techniques of genetic intervention or manipulation intended for the introduction of genetic material in somatic cells by means of artificial techniques, for the purpose of correcting genetic defects or stimulating immune responses against the phenotypic expression of genetic defects or preventing their occurrence. 3. Requirements for Proposals for Genetic Intervention or Manipulation in Human Beings The following shall be submitted to CTNBio for evaluation: a. biosafety quality certificate of the laboratory or institution; b. description of the proposal, with answers to the topics listed; c. detailed experimental protocol, including the complete nucleotide sequence of the gene to be transferred and of the vector; d. documentation demonstrating approval by the Research Ethics Internal Committees as set forth by Resolution No. 196/96 of the National Health Council, including documents of Free Informed Consent signed by the research subject, pursuant to the aforementioned resolution; e. the resumes of the investigators, especially informing any previous experience with genetic intervention or manipulation in human beings. 4. Specific Topics for Proposals for Genetic Intervention or Manipulation in Human Beings 4.1. Purposes and Strategy of the Proposal 4.1.1. Genetic intervention for Therapeutic Purposes 4.1.1.1. Why is the disease selected for treatment through genetic intervention in human beings is a good candidate for this treatment? 4.1.1.2. Describe the natural course of the disease selected for treatment. Are there objective criteria to quantify the activity and severity of the disease? Will the knowledge of the clinical evolution of the disease enable an accurate evaluation of the efficiency of the genetic intervention in human beings? 4.1.1.3. Was the protocol prepared to prevent the disease manifestations, prevent the disease progression after the appearance of the early symptoms to revert the disease manifestations in seriously ill patients? 4.1.1.4. Are there alternative therapies? What are the advantages and disadvantages compared with the genetic intervention in human beings? 4.1.1.5. Is there any experience of genetic intervention in human beings for this disease in other countries? In affirmative case, present the literature in that regard. 4.1.2. Genetic intervention for Other Purposes 4.1.2.1. What is the purpose of the protocol of genetic intervention? 4.1.2.2. Which cells will be the target of the genetic intervention? Why is the genetic intervention necessary? 4.1.2.3. Are there alternative methodologies? What are the advantages and disadvantages compared with the intervention? 4.2. Experimental Design, Expected Risks and Benefits 4.2.1. Structure and Characteristics of the Biological System Present a complete description of the methods and reactants to be used in the genetic intervention and the strategic reason for their use. Cover specifically the following topics: 4.2.1.1. In case of gene transfer, what is the structure of the cloned DNA to be used? 4.2.1.1.1. Describe the origin of the gene (genomic or DNA), the vehicle and form of the evidence that the material to be transferred corresponds to the intended gene transfer. Provide the complete nucleotide sequence, a detailed map of the construction and (incomplete) 4.2.1.1.2. What regulating elements are present in the construction (e.g. promoters, enhancers, polyadenylation sites, replication origins, etc.). What is the source of those elements? Summarize what is known about the regulating character of each element. Is the gene to be transferred potentially oncogenic? In affirmative case, what are the risks involved and what measures may be taken to reduce those risks? 4.2.1.1.3. Summarize the steps of the process to obtain the construction. 4.2.1.2. What is the structure of the material that will be administrated to the patient and how will it be administrated? 4.2.1.2.1. Describe the preparation, structure and composition of the materials that will be administrated to the patient or used to treat the patient’s cells: 4.2.1.2.1.1. In case of DNA, what is the pureness (both in terms of being a single molecular species and in terms of contamination with proteins, carbohydrates, lipids, etc.). What are the tests used to stimulate said pureness and what is its sensitivity? 4.2.1.2.1.2. In case of a virus, how was it prepared from the DNA construction? In what cells were the viruses grown? What medium and serum were used? How was the virus purification made? What is its pureness structure and level? What measures were taken (and how efficient are they) to detect the presence of contamination by other viruses, DNAs, RNAs and/or proteins? 4.2.1.2.1.3. In case that co-cultivation was used, what cells were used? What measures were taken (and how efficient are they) to detect the presence of any contamination? 4.2.1.2.2. Describe any other material that will be used in the preparation of the inoculum. For example, if a viral vector is being used, what is the nature of the helper virus? If other carrier particles are used, what is their nature? 4.2.2. Pre-Clinical Studies, Including Risk Survey Studies Describe results of trials in cell cultures or experimental animals demonstrating the safety, efficiency and feasibility of the proposed procedures. Explain why the experimental model chosen is the most appropriate one. 4.2.2.1. Gene transfer system 4.2.2.1.1. What are the target cells for the gene transfer? What cells will be treated ex vivo and reinserted in the patient? How will the selection of the target cells that will receive the transferred DNA will be made? How will the characterization of the cells will be made before and after treatment? What are the theoretical and practical data that enable to assume that only the target cells will receive the genetic material? 4.2.2.1.2. What is the efficiency of the gene transfer system? What is the expected percentage of target cells that will contain the transferred DNA? 4.2.2.1.3. How will the structure of the transferred sequences be monitored and what is the sensitivity of the analysis? Is the transferred DNA extra-chromosomal or integrated? May the transferred DNA suffer rearrangements? 4.2.2.1.4. How many copies of the transferred DNA are expected to be present per cell? What is the stability of the transferred DNA? 4.2.2.2. Gene Transfer and Expression in Terms of Persistence and Structure Stability 4.2.2.2.1. What models of tissue culture and experimental animals were used in laboratory studies to evaluate the in vitro and in vivo efficiency of the gene transfer system? What are the similarities and differences of those models compared with the proposed gene transfer to human beings? 4.2.2.2.2. What is the estimated minimum level of gene transfer and/or expression required for successful gene transfer? How was that level determined? 4.2.2.2.3. Explain in details the pre-clinical trials that demonstrate the efficiency of the transfer system in terms of minimum levels required for the gene transfer. 4.2.2.2.4. Does the integrated DNA modify the expression of other genes? How was that determined? 4.2.2.2.5. In what percentage of the cells that received the transferred DNA does gene expression occur? Is the product of the transferred gene biologically active? What proportion of normal activity is derived from the transferred gene? How was that determined? 4.2.2.2.6. Is the transferred gene expressed in any cells other than the target cells? How was that determined? 4.2.2.3. Retroviruses-Based Transfer Systems 4.2.2.3.1. What cellular types will be infected by the retroviral vector? Is the production of viral particles expected to occur? 4.2.2.3.2. How stable are the retroviral vector and the resulting provirus in terms of deletion, rearrangements, recombination and mutation? What information is available about the risk of recombination with endogenous retroviruses or other viruses that may be present in the patient’s cells? 4.2.2.3.3. Is there any evidence that the gene transfer may have any adverse effects (e.g. development of neoplasias, harmful mutations, regeneration of infectious particles, immune responses, etc.)? What precautions will be taken to minimize the pathogenicity of the retroviral vector? What pre-clinical trials were conducted to estimate said pathogenicity? 4.2.2.3.4. Is there any trial evidence that the vector may penetrate untreated cells, especially germination cells? What is the sensitivity of those analyses? 4.2.2.3.5. Was the gene transfer protocol for human beings tested in non-human primates or other laboratory animals? Specifically, is there any evidence of recombination of the retroviral vector with endogenous retroviruses or other viral sequences present in those animals? 4.2.2.4. Non-Retroviral Gene Transfer Systems 4.2.2.4.1. What animal trials were conducted to determine if there is any risk of undesirable or harmful consequences of the gene therapy protocol (including insertion of DNA in non-target cells, especially germination cells)? For how long were the animals studied after the treatment? What other biosafety trials were conducted? 4.2.3. Clinical Procedures, Including Patients Monitoring Describe the treatment that will be administrated to the patients and the diagnosis methods that will be used to monitor the response to treatment. Describe any previous clinical trials with the same or similar methods. Specifically, answer: 4.2.3.1. Will any patient’s cells be removed for ex vivo treatment? Describe the types and number of the cells and the intervals at which they will be removed. 4.2.3.2. Will the patients be treated to eliminate or reduce the number of unmodified target cells (e.g. radiation or chemotherapy)? 4.2.3.3. What treated cells (or combinations vector/DNA) will be administrated to the patients? How will the administration be made? What is the volume to be used? Will the treatment be single or multiple? What is the interval between treatments? 4.2.3.4. How will the transfer and expression of the gene in the patient’s cells be determined? Will the expression be examined in non-target cells? 4.2.3.5. What studies will be conducted to evaluate the presence and effects of contaminants? 4.2.3.6. What are the clinical endpoints of the study? Will there be any quantitative measurements to evaluate the natural history of the disease? How will the clinical follow- up of the patients be made? 4.2.3.7. What are the expectations in relation to the major benefic or adverse effects of the gene transfer? What steps will be taken to prevent or revert adverse reactions, in case they occur? 4.2.3.8. In case that a treated patient dies, what post-mortem studies will be conducted? 4.2.4. Public Health Considerations Discuss the possible risk of gene transfer to other persons besides the patients. Especially, answer the following questions: 4.2.4.1. Is there any risk to the public health? 4.2.4.2. Is there any possibility that the transferred DNA will spread from the patients to other persons or to the environment? 4.2.4.3. What precautions will be taken to avoid spreading? 4.2.4.4. What measures will be taken to minimize the risk to the public health? 4.2.4.5. Given potential risks to the progeny of the patients, including vertical transmissions, will contraceptive measures be taken? 4.2.5. Qualification of the Researchers and Appropriateness of the Clinical and Laboratory Facilities Describe the training and experience of the team. Describe the clinical and laboratory facilities that will be used. Specifically, answer the following questions: 4.2.5.1. Describe the facilities where the materials to be used in the genetic intervention will be prepared, including environmental conditions for occasional manipulation of ex- vivo cells. 4.2.5.2. What professionals will be involved in the pre-clinical and clinical trials and what are their qualifications? Include resumes. 4.2.5.3. In what hospital or medical office will the genetic intervention be made? What resources are especially important for the study proposed? Will the patients occupy regular beds or will they be isolated? Where will the patients reside during the follow-up period after the genetic intervention? 4.3. Patients Selection The patients selection criteria shall comply with the rules of Resolution No. 196/96 of the National Health Council. Estimate the number of patients involved in the study. Describe the procedures of selection of the patients. Specifically, answer the following topics: 4.3.1. How many patients will be treated? 4.3.2. How many candidates to genetic intervention may be identified per year? 4.3.3. What is the method of patients screening? 4.3.4. What are the selection criteria of the potential patients? 4.3.5. In case that there are more candidates to the genetic intervention than vacancies, what criteria will be used to select the patients?