III.1

Cellular and Gene Therapy for Cardiac Disease: FDA Product and Pre-Clinical Regulatory Considerations

A. Introduction

Heart failure and ischemic heart damage caused by coronary artery blockage are examples of cardiac diseases currently being evaluated for treatment with cellular and gene therapy products (CGTs) in clinical trials. The therapeutic goal of a CGT is usually the repair and regeneration of damaged cardiac tissue, but in some instances CGTs are also used to reduce damaging inflammatory responses. Examples of cell types that may be used to treat cardiac disease include adult bone marrow-derived products (hematopoietic progenitor cells, endothelial progenitor cells, mesenchymal stromal cells); tissue-derived (muscle, cardiac, adipose) progenitor cell products; cardiac cells derived from pluripotent cells; and combination products (e.g., cellular products with scaffold). Gene therapy constructs can be used to transfect cells ex vivo prior to their transplantation into a subject, or constructs can be administered directly to the subject. Examples of gene therapies used to treat cardiac disease include plasmids or viral vectors (e.g., adeno-associated virus and adenovirus) that express therapeutic transgenes.

Prior to the clinical use of a new CGT product, an Investigational New Drug (IND) application must be submitted to the Center for Biologics Evaluation and Research (CBER) within the Food and Drug Administration (FDA). The content of an IND submission needs to be organized into distinct sections containing Chemistry Manufacturing and Control (CMC) information, pharmacology and toxicology preclinical information, and a clinical protocol as described in Title 21 of the Code of Federal Regulations (CFR), section 312.23. An interdisciplinary team of CBER reviewers will review the IND application within 30 days of submission, according to the timelines established in the Prescription Drug User Fee Act. Consult reviewers from other FDA Centers may also help with a review if needed. The FDA’s primary objectives in reviewing an IND are to assure the safety and rights of subjects and in Phase 2 and 3 to help assure that the quality of the scientific evaluation (characterization) of drugs is adequate to permit an evaluation of the drug’s effectiveness and safety, 21 CFR 312.22(a). To aid in the preparation of a cellular therapy IND and clinical study for cardiac disease, CBER issued a guidance document entitled “Guidance for Industry: Cellular Therapy for Cardiac Disease”, that provides recommendations on the design of preclinical and clinical studies, and the Chemistry, Manufacturing, and Control (CMC) information to include in an IND submission (1). Investigators are also encouraged to contact FDA/CBER for scientific and regulatory advice in the early stages of CGT product development. A Pre-IND meeting with FDA can be requested to obtain answers to product specific CMC, preclinical, and clinical protocol questions.

CGT products intended to treat cardiac disease are evolving rapidly and the number of new IND submissions FDA will receive is expected to continue to increase with the emergence of new regenerative medicine products. This chapter will outline the current expectations for product manufacturing controls for investigational CGT products, general product testing requirements and expectations, and general considerations for preclinical study designs for CGT products intended to treat cardiac disease.

B. Chemistry, Manufacturing and Controls (CMC) Regulatory Requirements

The product design, manufacturing process, and clinical application of a CGT product for cardiac disease determine the regulatory concerns FDA will focus on during the review of a new IND application. For example, if a cell source is allogeneic then donor eligibility will need to be determined before the cells can be used in a clinical trial. If a CGT product requires a delivery device for administration, then product-device compatibility and device suitability need to be evaluated. If source cells are expanded and then stored in a cell bank, then the banked cells will need to be tested for adventitious agents. Depending on their design and expected mode of action, CGT products may require novel tests to address product quality and safety concerns. For more detailed descriptions of the CMC information that may be included in IND submissions, refer to the gene therapy (GT) and cell therapy (CT) guidances for IND content (2,3).

Regardless of the cell source and the subsequent protocols used during the manufacturing process, the FDA applies the same scientific principles and regulations when reviewing the CMC features of any CGT product. Some of the most relevant regulations applicable to CGT and Cell/Device Combination Products for cardiac disease can be found in the following sections of the Code of Federal Regulations (CFR) Title 21, Food and Drug Administration:

• Current Good Manufacturing Practice (cGMPs) – 21 CFR Parts 210 and 211
• Quality System Regulations – 21 CFR Part 820 (for Medical Devices)
• Biologics – 21 CFR 600-680
• Human Cells, Tissues, and Cellular and Tissue-Based Products – 21 CFR 1271 (includes Donor Eligibility and Current Good Tissue Practice)
• IND Content and Format – 21 CFR 312.23

Allogeneic donor eligibility determination

Allogeneic cells from a single donor can be expanded into cell banks containing a large quantity of cells capable of treating multiple subjects, or they may be used to treat single subjects. Some potential allogeneic cell sources for CGT clinical trials for cardiac disease include bone marrow-derived mesenchymal stromal cells, hematopoietic stem cells, cardiac progenitor cells isolated from heart biopsies, and cells derived from embryonic stem cells or induced pluripotent stem cells.

To prevent the spread of communicable disease, donors used for source materials of allogeneic cell-based products are subject to the donor eligibility testing and screening requirements for Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps) as required in 21 CFR 1271, subpart C. The communicable diseases that HCT/P donors must be screened and tested for, and more detailed recommendations for performing donor eligibility determination are provided in the FDA Guidance: Eligibility Determination for Donors of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps) (4). Donors are deemed eligible if they test negative for communicable diseases, have no disease symptoms, test negative for risk factors identified during donor screening. A designated person who has the appropriate training and adequate knowledge of relevant Federal regulations and guidance must determine the eligibility of a cell or tissue donor. A summary of the records used to make the donor eligibility determination, a distinct label for tracking, and an eligibility statement must accompany an HCT/P when it is distributed. It is critically important to determine donor eligibility prior to product manufacture, since material obtained from ineligible donors cannot be used in clinical trials. Note that cells and tissues isolated for the manufacture of an autologous CGT product are exempt from donor testing requirements.

Control of the manufacturing process

The amount of CMC information required for regulatory evaluation increases as CGT clinical trials proceed from early Phase 1 studies to licensure (Fig. 1). For a Phase 1 study, the information for CMC evaluation will include preliminary product characterization, documentation of the manufacturing process, identification of lot release specifications, and initial stability tests. Depending on the manufacturing process, safety data that documents sufficient depletion of potentially tumorigenic cells (for example undifferentiated pluripotent cells), removal of toxic residuals, or absence of adventitious viruses may be required before a Phase 1 study is allowed to proceed. A description of the shipping process and a demonstration that the product is stable under the proposed shipping conditions will be included. Product labeling procedures that ensure segregation and tracking during the manufacturing process need to be described. Documentation needs to be submitted to the FDA that demonstrates there are appropriate controls and record keeping for monitoring manufacturing consistency, product quality, and ensuring freedom from microbial contamination. A detailed description of the administration and preparation of the final product needs to be submitted for review by the FDA. The route of administration, the volume and concentration of cells, a description of the injection device, and product handling procedures will also accompany a Phase 1 IND submission. Pre-IND interactions with FDA to identify potential CMC issues are highly recommended.

Manufacturers need to strive to use cGMPs which are defined as “a set of current, scientifically sound methods, practices or principles that are implemented and documented during product development and production to ensure consistent manufacture of safe, pure and potent products”. Products that are being used in Phase 1 clinical trials are not required to comply with the cGMPs described in 21 CFR 211. Instead manufacturers may refer to the “Guidance for Industry: CGMP for Phase 1 Investigational Drugs” to help them establish an appropriate manufacturing environment and implement adequate process controls (5).

When a product has progressed to a Phase 3 clinical trial, scale-up manufacturing issues need to have been addressed and a potency assay must be in place (Fig. 1). CGT manufacturers need to strive to control product variability by using processes that are reproducible. By the time of license application, the manufacturing procedures need to be fixed and specifications refined. The critical steps of the manufacturing process are monitored by testing to control variability, and the manufacturing process is validated.

Qualification of manufacturing reagents

Depending on the CGT product, the manufacturing process may include cell isolation and purification, cell expansion in culture, genetic modification, and assembly of cell-scaffold constructs. Reagents used during manufacturing that are animal derived such as antibodies, cytokines, growth factors, culture media, and serum have the potential for contaminating the product with adventitious agents. For example, monoclonal antibodies used for the isolation of cells have the potential to introduce adventitious viruses, therefore the antibodies need to be purified using validated viral clearance methods per the guidance “Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use” (6). If fetal bovine serum is used in the culture media, then documentation is required to show it was obtained from sources that minimize the risk of contamination from the agent of bovine spongiform encephalopathy (BSE) and other potential adventitious agents (7). If cells are grown on non-human feeder cell layers, there is a potential for retroviral and zoonotic contamination, and therefore the CGT product would be classified as a xenotransplantation product (8).

Whenever they are available, manufacturers are advised to use clinical grade or FDA-approved reagents obtained from reliable sources, which provide reagent performance and safety documentation. If research grade reagents are used during the manufacturing process, certificates of analyses will be needed to verify the quality of the reagent. Since cell-based products cannot undergo a sterilization process, manufacturers of recombinant, human or animal-derived reagents are advised to supply documentation showing the reagents have been tested for the presence of viral, bacterial, fungal, endotoxin, and mycoplasma contamination. If this information is not available, the CGT manufacturer investigator may be required to perform sterility and adventitious agent testing on the reagent in question.

Characterization of cell banks

In some cases it may be advantageous for CGT products to be stored in a cell bank at an intermediate stage of processing. A cell bank is comprised of a population of cells isolated, purified, and expanded from a source material under defined conditions. The cells are usually stably stored in liquid nitrogen at an intermediate stage of processing until they are needed for manufacture of a product lot. To enable consistent manufacturing, it is advised that cellular features such as passage number, expansion potential, viability, and cell source are documented. Cytogenetic analysis is advised to document the genetic stability of banked cells. Genetic fingerprinting using techniques such as short tandem repeat (STR) analysis may be used to distinguish one cell bank or cell line from another. Biological and immunological activity assays can also be used to evaluate cellular health and function. Depending on the final product, cell banks may need to be tested for their ability to efficiently differentiate into desired cell types.

Requirements for testing cell banks for adventitious virus and bacterial contamination are based on a risk versus benefit analysis that is dependent on the reagents used during manufacturing, the source of the cells, and the clinical indication that they will be used to treat (9, 10). Potential tests include assays for sterility, mycoplasma, human pathogens, adventitious viruses in vitro and in vivo, and retroviral testing if cells or reagents are exposed to animal materials that may contain retroviruses.

Populations of cells that have been grown in culture, regardless of their source, are likely to have some cell marker and cell differentiation heterogeneity. This inherent heterogeneity is acceptable, but recording the degree and types of cellular heterogeneity is advised throughout the clinical study. In addition, it is advised that a testing program that measures critical cellular attributes is established to assess the stability of the cell bank during the intended storage period.

During the lifetime of a CGT product, cell banks may be generated from different donors. Genetic differences between donors have the potential for introducing variations in cellular potency, immunogenicity and tumorigenic potential. Product variations can complicate the interpretation of both safety and efficacy data. Thus identification of methods that can demonstrate cell bank and cellular comparability is advised. (11, 12).

Product compatibility with catheter

Delivery of CGT products for cardiac disease often relies on specially designed injection devices that allow the delivery of the product to specific anatomical locations such as ischemic myocardium or a coronary artery. Before the start of a clinical trial it is necessary to demonstrate that the delivery of the CGT product is compatible with the delivery device. Acceptable recovery and cell viability can be analyzed by setting up mock infusions or injections through the proposed delivery device under conditions that simulate the clinical use as nearly as possible. Cells and GT vectors may adsorb to syringes or catheters, so the percent of recovery after passage through the device needs to be measured. Likewise, cells may be sheared during delivery. For these reasons cell viability is measured before and after passage through the device. The results of these bench tests are present in the IND submission and may be useful in planning the dose of the product that will be administered to the subject.

Lot release specifications

Manufacturers of biological products licensed under authority of Section 351 of the Public Health Service Act must ensure that each lot of their product(s) is safe, pure and potent prior to commercial distribution (also known as lot release). General lot release testing requirements for the sterility, identity, purity, and potency of Biologics are described in 21 CFR Part 610. Specific testing that may be needed to meet these requirements will vary with the product, but for a cellular product it may include cell viability, cell surface marker analysis, and phenotypic characterization.

Lot release testing for CGT products is complicated by several factors. Since many of these products are novel, there are usually no standard acceptance criteria that can be used. The complexity of the final product and the variability of the starting materials make it difficult to manufacture products that meet narrowly defined specifications. CGT products are often produced in small lots that limit the material available for testing. Finally, since many CGT products are processed and then administered as soon as possible there may be very limited time available to perform testing and review results. It is advisable to discuss lot release criteria with FDA as early as possible during the development of a proposed CT.

Microbial testing

Many CGT products will be administered within hours of their preparation. Therefore results from a culture based sterility test would not be available prior to product administration. In these cases, a Gram stain is performed before issuing the product. In addition, sterility cultures are initiated on samples of the final product, results monitored, and positive results reported promptly. A comprehensive action plan that includes physician and patient notification in the event of a positive sterility test are submitted with each CGT IND. For cultured cellular products, a test for mycoplasma is performed at the end of the culture period. To maximize detection of potential contamination, we recommend obtaining samples containing both culture supernatants and cells prior to final wash procedures.

Purity

Purity is defined as meaning relative freedom from extraneous matter and freedom from pyrogenic substances. Potential impurities that may be found in CGT products include endotoxin, residual solvents, antibiotics, animal products such as fetal calf serum, growth factors, and undesired cell types. Acceptable levels of all types of impurities need to be identified and methods to remove excess amounts of impurities are investigated. Methods used to remove or reduce impurities and/or tests to measure residual levels need to be validated by Phase 3. For cell-based therapies, the manufacturer needs to define the product by performing a quantitative assessment of unintended cell types. The type(s) of purity testing that are developed and performed on CGT products for cardiac repair will depend on the nature of the product, the manufacturing process, and the ancillary materials used. For example, if immunogenic or toxic materials are used in collection or processing, their removal by washing or other steps must be demonstrated. Alternatively, testing must be developed and performed on each lot to demonstrate safe levels for administration.

Identity testing

Identity testing is needed to distinguish the final product from other products produced in the same facility and to demonstrate that the product matches the package labels. Identity may be established either through physical or chemical characteristics, inspection, or specific bioactivity tests. If the product is comprised of multiple components (e.g. cell lines), then the test method(s) needs to identify each component.

Potency testing

Potency is defined as the specific ability or capacity of a CGT product, as indicated by appropriate laboratory tests, to affect a given result (13). Therefore, efforts to determine the mechanism(s) of action are needed during product development and pre-clinical testing in order to design an appropriate potency assay. A potency assay has to be in place by the beginning of a Phase 3 clinical trial and must be validated before a Biologics License Application is approved. A CGT product potency assay could measure the expression of a transgene/protein, a desired cellular activity, or the structural integrity of a cell-scaffold product. Potency assays may also consist of correlative assays that are predictive of cellular behaviors, such as engraftment or cell survival potential. Depending on the nature of the product, multiple assays may be needed to demonstrate potency.

Since many CGT products are administered shortly after manufacture, their potency assays must be designed to take a minimal amount of time because potency tests results must be available before the product is released.

Gene therapy products

Gene therapies are products that mediate their effects by transcription and/or translation of transferred genetic material and/or by integrating into the host genome and that are administered as nucleic acids, viruses, or genetically engineered microorganisms (14). The products may be used to modify cells in vivo or transferred to cells ex vivo prior to administration to the recipient. Examples of GT vectors include adeno-associated virus, adenovirus, herpes simplex virus, lentivirus-HIV based vectors, retroviruses and plasmids.

Characterization of GT products is required to obtain data on which to base lot release specifications, to show comparability after manufacturing changes, and to demonstrate lot-to-lot consistency (3). A detailed description of the derivation of the construct, a vector diagram, and sequence analysis of the vector must be submitted with a Phase 1 IND application. Master and working banks nee to be tested for replication competent viruses, sterility, identity, genetic integrity, adventitious agents, and transgene expression. If autologous or allogeneic cells are transduced ex vivo, a detailed description of the cell source, cell culture, and transfection procedures needs to be submitted for review. GT final product characterization needs to include the measurement of transgene expression. In addition, a potency assay is developed by Phase 3 that consists of in vivo or in vitro tests that measure an appropriate biological activity. One of the primary goals of product characterization is to define the critical quality attributes of the product that can be linked to the clinical safety and efficacy data obtained in the clinical trials.

GT study subjects exposed to gene transfer technology, especially those designed to stably integrate, may be at risk of delayed adverse events as a consequence of persistent biological activity of the genetic material. A long-term follow-up plan may be required to detect potential GT-related delayed adverse events (14).

Combination products

When medical products are comprised of multiple components that would individually be regulated under different regulatory pathways (i.e, device, drug, or biologic), they are classified as Combination Products. Examples of combination products for cardiac indications include cardiac patches made of cells seeded on biodegradable material, drug eluting stents, and the percutaneous intracoronary or intracardiac injection of cells, or other biologics.

Combination products are defined in 21 CFR 3.2(e) as either

1. A product comprised of two or more regulated components, i.e., drug/device, biologic/device, drug/biologic, or drug/device/biologic, that are physically, chemically, or otherwise combined or mixed and produced as a single entity;
2. Two or more separate products packaged together in a single package or as a unit and comprised of drug and device products, device and biological products, or biological and drug products;
3. A drug, device, or biological product packaged separately that according to its investigational plan or proposed labeling is intended for use only with an approved individually specified drug, device, or biological product where both are required to achieve the intended use, indication, or effect and where upon approval of the proposed product the labeling of the approved product would need to be changed, e.g., to reflect a change in intended use, dosage form, strength, route of administration, or significant change in dose; or
4. Any investigational drug, device, or biological product packaged separately that according to its proposed labeling is for use only with another individually specified investigational drug, device, or biological product where both are required to achieve the intended use, indication, or effect.

Combination product primary review responsibility

The FDA center that has primary responsibility for the review of a combination product is determined based on its primary mode of action (if possible). A combination product of a CGT product combined with a scaffold or a dedicated delivery system (i.e., cardiac catheters) would generally be assigned to CBER as the primary Center for review since the CGT product is providing the primary therapeutic mode of action. A single investigational application would generally be sufficient with a concurrent consult review performed by the Center for Devices and Radiological Health (CDRH).

If it is unclear which component is responsible for the primary mode of action, a Sponsor can submit a request for designation and provide FDA with information that can be used to help make a decision about which component provides the primary intended activity (link to FDA reference). Within 60 days FDA issues a letter of designation specifying the Center that will have primary review responsibility. Consult or collaborative reviews for secondary components are performed by Centers with component expertise, as needed. Depending on its designation and its primary mode of action, the combination product may be reviewed using Biologic License Application (BLA), PreMarket Approval (PMA), 510(k), or New Drug Application (NDA) processes.

CMC Evaluation of cell-scaffold medical products

Investigational products that combine cells with a scaffold construct are very challenging to reproducibly manufacture and because of their complexity, are also difficult to evaluate. Like other CGT products, each lot of a cell-scaffold product must be tested to determine if it meets pre-defined lot release criteria (15). Depending on the product, lot release criteria may include testing for sterility, mycoplasma, pyrogenicity/endotoxin, scaffold characteristics, cellular viability, identity, purity, and potency. Ideally, as much testing needs to be done on the final cell-scaffold product as feasible, but in some cases lot release testing may have to be done on individual components because it is impractical or impossible to perform on the final product. For example, scaffold structural integrity and cell marker analyses may need to be done on individual components prior to assembly. Advances in imaging techniques that can non-destructively measure viability, structural integrity, and biological activity of a fully formed cell-scaffold product are being developed. Some examples include advanced non-invasive optical methods such as Raman spectroscopy, detection of fluorescent metabolites, and optical coherence tomography.

C. Preclinical Testing Program for Cardiac Disease

Preclinical safety evaluation is required for all new investigational products prior to their use in clinical trials, 21 CFR 312.23(a)(8). Therefore, the inclusion of preclinical data that sufficiently establish a scientific rationale and demonstrate an acceptable safety profile of the CGT investigational product for clinical investigation is an important component of the IND regulatory submission. The diversity in the type of CGT products, the route of product administration, the delivery catheter used, and the clinical indication itself, must all be considered before starting a clinical trial. Each of these factors can present a different spectrum of biologic activities and safety concerns, and thus be a potential regulatory challenge. Therefore, submission of a well-designed, well-executed preclinical testing program is essential to a successful filing of an IND.

Although the design of a preclinical testing program usually depends on the previously cited factors (i.e., case-by-case), the overall objectives for all preclinical programs is the same: 1) establishment of a biological response of the test system (animal or cells) to the product; 2) determination of the in vivo durability of that response (the length of time needed to assess the effect of the investigational product on the heart and the durability of that effect); 3) recommendation of a safe starting dose level, dosing regimen, and dose-escalation scheme in the clinical trial; and 4) identification of any potential toxicities of the investigational product.

In order to address these objectives, the preclinical program will incorporate a tiered approach, consisting of the conduct of proof-of-concept (POC) studies to provide data to support product activity and mechanism of action (MOA), followed by pilot studies to explore key parameters (such as animal species/models selection, time points for assessment of in vivo cell fate for CT products and tissue biodistribution for GT products, study duration, etc) that can help to design the pivotal preclinical studies. For a more comprehensive summary regarding the preclinical assessment of CGT products, refer to the document, titled at: Guidance for Industry: Preclinical Assessment of Investigational Cellular and Gene Therapy Products

POC studies in vitro and in vivo

A primary objective of the POC studies is to establish the feasibility and rationale for delivery of an investigational CGT product to the target patient population. Data generated from POC studies need to address the following: 1) the pharmacologically effective dose range (i.e., the minimally effective dose and optimal biologic dose); 2) optimization of the clinical formulation (e.g., formulation buffer, scaffold composition); 3) establishment of the product administration procedure (e.g., target anatomic site(s) of product administration, number of injections/site, volume/injection); 4) optimization of the timing of product administration relative to onset of disease/injury (e.g., the timing of administration post-MI); 5) characterization of the proposed MOA or hypothesized biological activities of the investigational product; and 6) identification of relevant animal species/model(s) to use in the preclinical pivotal studies.

Preclinical in vitro testing can also provide an understanding of the MOA and the overall biological activity of the investigational CGT product. Examples include growth factor secretion, immunological response profile, cardiac lineage differentiation of the cells, and the level of transgene expression. However, in vitro testing alone is rarely sufficient to provide information that can reliably predict the physiologic effects of the product following in vivo administration. For in vivo preclinical testing, an animal model that recapitulates some aspect of the clinical disease/injury (e.g., etiology, pathophysiology) can be used to allow for characterization of morphological changes in conjunction with any observable functional changes. Collectively, in vitro and in vivo POC studies conducted in the early stages of a preclinical testing program serve to establish the rationale for, and the feasibility of, the study procedures that will be used in the clinical trial and can help inform the design of the pivotal animal studies.

Considerations for selecting animal species and animal models of cardiac disease/injury

A preclinical program for an investigational CGT product for a cardiac disease does not necessarily require the use of multiple animal species or the use of a non-human primate (NHP) species. However, scientific justification needs to be provided for the choice of animal species. Although safety and activity of the investigational CGT product can possibly be evaluated in one animal species, other contributory factors, such as the source of the product or the route of administration (ROA), may result in the need to test in more than one species. Some factors that need to be considered when determining the most relevant species include: 1) comparability of physiology, electrophysiology, and anatomy of the animal heart to the human heart, 2) feasibility of using the intended clinical catheter delivery system, 3) immune tolerance to a human CT product, 4) permissiveness/susceptibility of an animal species to infection by the viral vector for a GT product, and 5) comparable activity of a human transgene in animals and humans. Data generated from in vitro and in vivo pilot studies, as well as applicable data reported in the published scientific literature, need to be provided to support the biological relevancy of the selected animal species.

Of note is that administration of a product consisting of human cells into immune-competent animals can potentially result in product rejection. This response prevents adequate evaluation of the potential activity and safety of the human cellular product. This cross-species immunogenicity may necessitate modification of the animal in order to create an in vivo immune-tolerant niche for the human cells. Commonly used options include: 1) human cells administered into genetically immunodeficient rodents and 2) human cells administered into immune-competent animals exposed to an immunosuppressive regimen. However, each option has disadvantages. The major limitations of using immunodeficient rodents are the significant differences in the anatomy, physiology, and electrophysiology between a human and a rodent heart. The major limitations of using animals treated with immunosuppressive agents include: 1) the difficulty in achieving a consistent level of immunosuppression, 2) the challenge of conducting a long-term study, and 3) possible effects of the immunosuppressive agents on the cells themselves, making it difficult to discriminate immunosuppression-related toxicity from CT product toxicity.

The administration of analogous cellular products (i.e., CT products derived from the animal species used for testing) in the animals is another potential consideration. However, when preclinical studies are performed using an analogous CT product, differences in the biological activity, molecular regulatory mechanism, and impurities/contaminants between the human and the animal product may exist. Therefore, if this pathway is used, all differences in manufacturing and in the final product characteristics (i.e., phenotype and function) between the CT product administered to the animals and the CT product proposed for clinical use needs to be thoroughly documented in the regulatory submission.

For a GT product, the expressed transgene can also be a factor when determining a relevant animal species for preclinical testing. When the clinical transgene product is biologically active only in humans and in a single or a restricted number of animal species, the use of these species to evaluate the safety and activity of the clinical GT product is an important consideration. In this situation, use of a GT product construct identical to the intended clinical construct, but expressing the analogous animal transgene may be an option for the preclinical studies. As for a CT analogous product, additional characterization of the GT product administered to the animals needs to be thoroughly documented in the regulatory submission in order to determine similarities and differences between the preclinical and clinical GT products (16).

Preclinical studies performed in animal models of disease/injury are important in order to better define the risk-benefit ratio associated with investigational CGT products. Due to some properties common to many CGT products, such as prolonged product persistence in vivo; a complex MOA; and invasive ROAs, the effects of the local microenvironment (e.g., the milieu associated with ischemic cardiac tissue) and the clinical indication (e.g., acute vs. chronic MI) can impact the activity and safety of the administered CGT product. Thus, a preclinical program that employs only healthy animals is not likely to provide sufficient insight into the overall risk profile of the CGT product. In addition, the use of disease/injury models provides a potential opportunity for identification of activity-risk biomarkers that can potentially be further evaluated in the clinical trial.

A comprehensive discussion of the similarities and differences between an animal model of cardiac disease and the target patient population is an important justification for use of a particular model in the preclinical studies. Such a discussion needs to include: 1) disease etiology, 2) disease pathophysiology, 3) stage of the disease, 4) disease severity and symptoms, and 5) relevant cardiovascular, biochemical, and hemodynamic parameters that can be measured in the animal model.

In addition, the potential limitations of animal models needs to be taken into consideration when designing and interpreting the preclinical studies. Examples of animal model limitations include: 1) the inherent variability of the model (e.g., the size of the induced MI), 2) limited historical/baseline data, 3) technical limitations due to the physiological and anatomical constraints of the model, 4) animal care issues, and 5) limited fidelity in modeling the pathophysiology of the human disease of interest. Each model has inherent strengths and weaknesses. Many times the models do not recapitulate all aspects of the disease/injury pathophysiology that is observed in humans, such as the complex pathophysiology of some congenital heart diseases. Therefore, careful consideration is critical to which model(s) may provide the best insight into the in vivo safety and activity profile of the CGT product that is relevant to the clinical situation.

Small animal models

Small animal species, primarily rodents, have been widely used in the assessment of preliminary activity and possible MOA for CGT products intended to treat MI and cardiomyopathy. The advantages of rodent species include: 1) a robust sample size, 2) availability of transgenic or knockout models, and 3) the ability to create surgically induced lesions in immunodeficient animals to study the intended human CT product. However, due to the differences in the intrinsic heart rate between humans and rodents, along with anatomical and physiologic constraints related mainly to body size, rodents are not well-suited for assessment of arrhythmogenicity, measurements of left ventricular function and volume, monitoring for catheter-mediated injury, replicating the planned clinical administration procedure, or the ability to administer the absolute human dose level and dose volume.

Large animal models

Given the limitations of the small animal species for evaluating some important parameters of the CGT products for cardiac disease, large animal models are commonly used in the preclinical development program. The cardiac physiology and anatomy of large animal species, such as pigs, sheep, and dogs, allow for use of the clinical delivery device system and administration of dose levels and volumes that approach the absolute human levels. Thus, extrapolation of the dosing parameters for targeted cardiac delivery can approach the actual planned clinical scenario. Myocardial ischemia can be induced in large animal models by ischemia/reperfusion, ameroid constriction, or bead embolization. However, the pathologies generated by these methods are not fully representative of atherosclerotic coronary disease, the predominant underlying major cause of chronic myocardial ischemia in humans. Even with such limitations, these induction procedures can model acute and chronic ischemic heart disease because they allow assessment of CGT products in an ischemic microenvironment, and in tissues injured as a consequence of the ischemia. To that end, large animal models of myocardial ischemia have been extensively used to evaluate the safety and activity of CGT products (17).

When choosing a method for creating myocardial ischemia in a large animal species to reflect the patient population, consideration needs to be given to: 1) the anatomy, size, structure, and distribution of the coronary artery, 2) the infarct size and location, 3) the level of left ventricle global impairment, and 4) the pattern of remodeling due to the MI.

The availability of non-ischemic heart failure large animal models is somewhat more limited than for ischemic injury. Reported models of non-ischemic heart failure that have been used to assess the safety and activity of CGT products include a porcine model of volume overload (18) and an ovine model of pacing-induced dilated cardiomyopathy (19).

If the clinical development will include both patients with ischemic heart failure and patients with non-ischemic heart failure, then a tiered approach using multiple heart failure models needs to be considered, in order to address questions of safety and activity of the CGT product for this broad clinical indication.

Toxicology studies

Toxicology studies are conducted in animals to evaluate the potential for local and/or systemic adverse effects when the CGT product is administered. Examples of potential adverse effects of CGT products intended to treat cardiac disease include: 1) altered cardiac function, 2) inflammatory or immune response in cardiac and/or non-cardiac tissues, 3) arrhythmogenicity, 4) thromboembolic phenomena, 5) ectopic tissue formation, and 6) tumorigenicity. Although healthy animals represent the standard test system employed to conduct traditional toxicology studies, study designs using animal models of disease/injury are frequently modified to incorporate important safety parameters that allow for assessment of the potential toxicity of an investigational CGT product (i.e., hybrid pharmacology-toxicology study design) for the treatment of cardiac disease. The overall design of the preclinical toxicology study will optimally mimic the proposed clinical trial design as closely as possible, to include: 1) administration of the intended clinical product; 2) the planned clinical dosing regimen; 3) the planned clinical ROA, device delivery system, and delivery procedure; and 4) CGT administration at a clinically relevant time point relative to onset of disease/injury (i.e., days/weeks following MI). The parameters incorporated in the definitive preclinical study design(s) will be guided by the data generated in the POC and pilot studies.

Preclinical toxicology studies will be conducted in compliance with Good Laboratory Practice (GLP), per 21 CFR Part 58. However, if the study is not conducted in compliance with these regulations, a brief statement citing the reason for the noncompliance, the areas of deviation, and whether the deviation(s) impacted the study outcome, will be included in the final study report. In addition, the facility conducting a non-GLP preclinical study that collects safety data will use a prospectively written protocol and maintain appropriate record keeping and documentation of all data. Oversight of the conduct of this study and the resulting final study report by an independent Quality Assurance unit/person is also recommended

.

Adequate numbers of animals

The number of animals necessary will vary depending on the complexity of the CGT product, the suspected safety concerns based on product biology or other data, the animal species/model used, and the device delivery system used. The number of animals included needs to allow for biologically interpretable results. In addition, if a high attrition rate is expected due to the severity of the induced cardiac lesion or to the immune status of the animal species, additional animals need to be added to the study to ensure that the desired number of evaluable animals can be followed to scheduled sacrifice (assuming no toxicity occurs).

Non-biased study design

Appropriate control groups need to be included to minimize bias when interpreting the study results. Examples of control groups include animals that: 1) do not receive the CGT product, 2) receive vehicle or scaffold only, and 3) undergo a sham procedure with no delivery of product or vehicle. If the pivotal study is performed in an animal model of disease/injury, and depending on the model, the complexity of the product, and the delivery procedure, then inclusion of groups of animals with normal hearts can be considered. Such groups may be useful to evaluate the effect of the product on healthy cardiac tissue and to distinguish adverse findings caused by the product or the delivery procedure from those associated with the model itself. Justification need to be provided for the control group(s) used.

The animals need to be randomly assigned to groups in a pre-specified manner, based on pre-specified parameters such as body weight and baseline cardiac lesion, in order to minimize the potential for bias in interpreting of study outcome.

Additional ways to minimize potential bias in data interpretation include assessment of data generated from cardiac parameters by an independent board-certified veterinary cardiologist who is blinded to treatment groups and evaluation of histopathology data by an independent board-certified veterinary pathologist who is blinded to the study groups.

Product administration

When feasible, the identical CGT product that will be administered to the patient population will be used in the pivotal preclinical study. Similarities and differences in the manufacturing process and in product characterization specification between the CGT product lots used in the preclinical study and the lots that will be used clinically need to be identified and discussed in the IND submission. If there are significant differences, additional preclinical data may be needed.

A number of different ROA are used in cardiac CGT trials, namely: 1) direct, syringe-and-needle injection of CGT products into the exposed epicardium during concomitant thoracic surgery (e.g., during coronary artery bypass graft (CABG) surgery, transmyocardial laser revascularization (TMR) procedure, left ventricular assist device (LVAD) implantation), or via minimally-invasive thoracotomy; 2) infusion of CGT products into the coronary arterial system using a cardiac infusion catheter; 3) delivery of CGT products via a percutaneous transendocardial catheters; and 4) intravenous infusion. The ROA used to deliver the investigational CGT product in the pivotal preclinical study will optimally mimic the ROA that will be employed in the clinical setting to the greatest degree possible. The product delivery procedure used in the pivotal study will mimic the proposed clinical procedure as closely as possible, including but not limited to the use of the clinical delivery device, anatomic location of the injection site(s), number of injections, product concentration, volume of injection (total injectate and individual injection volume), injection/infusion rate, depth of injection, and other delivery parameters such as the number and duration of balloon inflation/deflation sequences when performing intracoronary administration.

Study duration

The preclinical safety study needs to be of sufficient duration to assess for both potential acute (i.e., within days of product administration) and chronic (i.e., within several weeks or months) toxicities that could be a result of the CGT product, the delivery procedure, or other factors. The POC studies, as well as providing some understanding of the in vivo cell fate for CT products and tissue biodistribution profile for GT products, can help justify the duration for the pivotal study. Depending on concerns due to specific attributes for some CT (e.g., embryonic stem cell-derived products) or GT (i.e., integrating vectors) products, the definitive preclinical studies may need to encompass extended periods of time.

Determination of a no-observed-adverse-effect-level (NOAEL)

Multiple dose levels of the investigational CGT product that bracket the proposed clinical dose level range need to be studied. Results obtained from POC studies will guide selection of the target dose levels to be administered. Data generated from the toxicology studies will potentially establish a NOAEL, which is the highest dose level at which no biologically or statistically significant increase in the severity or frequency of adverse findings in morphology, function, or other observation parameters (e.g., mild serum chemistry or hematology changes of no clinical significance) compared to appropriate concurrent controls, is observed. This determination will help justify selection of the starting dose level and subsequent dose-escalation scheme for the clinical trial.

Standard toxicology endpoints

Standard toxicology endpoints include mortality (with cause of death determined, if possible), clinical observations, body weights, physical examinations, food consumption/appetite, water consumption (as applicable), clinical pathology (serum chemistry, hematology, coagulation, urinalysis), organ weights, gross pathology, and histopathology.

Cardiac safety and functional endpoints

In addition to the standard toxicology endpoints, ‘cardiac disease’-dependent endpoints will also be evaluated. Cardiac markers (i.e., troponin I, CPK-MB) will be measured at baseline (pre-lesioning, post-lesioning/pre-dosing) and at multiple time points within the first week after product administration and at subsequent time intervals in order to monitor for possible adverse effects due to administration of the CGT product.

Cardiac rhythm needs to be monitored peri-procedure and at multiple time points post-dose. Serial EKGs, implantable loop recorders (ILRs), and telemetry devices have been used for monitoring cardiac rate and rhythm in large animal models for safety evaluation of CGT products. The advantages of using a continuous rhythm monitoring device (e.g., ILR) include: 1) all pre-specified events can be captured independent of an assessor being present, and the data are available for future downloading and analysis, and 2) the recorded data allow the pathologist to deduce a possible correlation of pathological lesions with electrophysiologic events, thus determining the significance and the potential cause of the lesions. If arrhythmogenesis is observed, additional large animal studies may be needed to explore factors that may contribute to this finding, such as the specific composition of the CGT product, the dose and volume of the CGT product administered, and the site of product injection (to determine whether the administration procedure results in injury to important cardiac structures such as the conduction system and heart valves).

Imaging techniques are often used in large animals to monitor the effect of a CGT product on cardiac physiology and function. The commonly used non-invasive techniques include echocardiography (ECHO) and magnetic resonance imaging (MRI) to evaluate cardiac chamber dimensions/volumes and regional and global myocardial function. The invasive techniques include cardiac catheterization to assess pressure/volume relationships. It is not expected that all of these techniques would be performed as part of the preclinical development program, but the rationale for selection of the technique(s) used will be provided. The monitoring frequency, with appropriate justification, will be pre-specified in the study protocol.

One of the primary advantages of animal studies is the ability to collect pathology data. Although the target site is the heart, comprehensive histopathology assessment needs to be performed by an independent veterinary pathologist who is blinded to treatment groups to evaluate potential cardiac and extra-cardiac organ toxicity. Analysis of cardiac pathology will include detailed sectioning of the heart, with extensive microscopic examination, including standard H&E and other applicable staining (e.g., Trichrome staining; 2,3,5-triphenyltetrazolium chloride staining (TTC)). Cardiac pathology analysis includes evaluation of: fibrosis, calcification, granulation tissue, inflammation, vascularity, myocarditis, pericarditis, necrosis, and tumor formation. For any lesions observed in the cardiac and peri-cardiac tissue, the frequency, severity, potential cause, and clinical significance need to be determined. Any potential product-related inflammatory/immune response, necrosis, and fibrosis needs to be further characterized, such as by immunohistochemistry, in an attempt to determine potential product attribution. Comprehensive gross pathology and histopathology assessments, as well as analysis of other applicable parameters (e.g., clinical pathology, cardiac biomarkers, vector presence), needs to be conducted for any animal that is found dead or sacrificed moribund (i.e., unscheduled death) to elucidate a possible cause of death.

D. Specific ‘Pharmacokinetic’ Considerations for CGT Products

As a consequence of their biologic attributes, CGT products administered in vivo are not subject to conventional chemical analyses; therefore, standard absorption, distribution, metabolism, and excretion (ADME) and pharmacokinetic testing techniques and profiles are not applicable. Although influenced by specifics of the CGT product and its ROA, these products have an inherent potential to distribute to sites other than to the target organ/tissue.

Evaluation of cell fate in vivo post administration

CT products are delivered into humans using various routes of administration. Thus an important aspect of the animal studies is the evaluation of cell fate of a CT product following administration. Cell fate can consist of identifying factors such as cell survival/engraftment, distribution in the body, differentiation, integration into host tissues, and uncontrolled growth. If long-term cell survival/engraftment is necessary for product efficacy, in vivo cell survival, anatomic engraftment, and biologic activity over prolonged periods of time post-administration are endpoints that need to be considered in the animals. For CT products that are delivered into the heart (left or right ventricle), the in vivo distribution profile needs to be assessed. Examples of techniques used include various imaging modalities, polymerase chain reaction (PCR) analysis, and immunohistochemistry. Depending on the differentiation status of the cells and the extent of manipulation the cells undergo before use, the level of differentiation following administration in the animals needs to be assessed. In addition, the potential for dysplasia or hyperplasia is also important to address, depending on the differentiation status of the CT product, the extent of cell manipulation employed during manufacturing, and the cell growth kinetic profile.

Evaluation of tissue biodistribution of GT products

GT products are delivered into humans using the same ROA as for CT products. Therefore, characterization of the vector biodistribution profile following administration in the animals is an important parameter to capture. These data determine vector presence in various biological samples, consisting of cardiac tissues, non-cardiac tissues (including gonads), and biological fluids. Analysis of the biological samples will be conducted at the molecular level, using a quantitative PCR (qPCR) assay to determine the number of vector copies per microgram of genomic DNA at specified time points after vector administration. Cardiac tissue collected for this purpose will include (as applicable) injected and non-injected sites, regions perfused by the infused vessel (particularly relevant for GT delivery via intracoronary infusion), and remote regions. An outline of the qPCR assay methodology and a recommended basic panel of tissues to collect and analyze can be found in the document ‘Guidance for Industry: Gene Therapy Clinical Trials – Observing Subjects for Delayed Adverse Events’ (14). In addition, the presence of vector sequence in a biological sample can result in further analysis to measure the transgene expression level in that particular tissue.

Determining the vector presence, persistence, and clearance profile in the various biological samples can help guide the pivotal animal study protocol design. Examples include selection of the dose levels, dosing schedule, data collection time points, and the animal sacrifice time points. The biodistribution data, coupled with other safety endpoints such as clinical pathology, histopathology, and cardiac-specific tests, help determine whether vector presence or gene expression correlates with any tissue-specific findings or effects in the animals.

Considerations for CGT product delivery systems

In many instances, a catheter delivery system is used to administer the CGT product. To assess the potential risks associated with these devices and the accompanying delivery procedure, the delivery device system and the delivery procedure used in the pivotal preclinical studies will be identical to the planned clinical device and administration procedure, to the extent possible. Additional preclinical studies may be necessary to adequately assess the safety of the clinical device and the delivery procedure. If an active regulatory submission, such as a Master File (MF), exists for the device, these data may also be of value. In these circumstances, a letter of cross-reference from the sponsor of the cited regulatory file(s) is needed. Published animal or human studies that used the clinical delivery device or a related device may also provide supportive safety data. Additional advice regarding evaluation of the performance of a delivery system for cardiac studies is contained in Section V of the FDA document ‘Guidance for Industry: Cellular Therapy for Cardiac Disease’ (1).

Considerations for CT products with implantable scaffolds

Some CT products are delivered using a scaffold construct. Preclinical study designs for these combination products need to take into account the following:

• The scaffold will be adequately characterized (as applicable) for composition, degradation profile, biomechanical performance, and biocompatibility (i.e., host response to the scaffold component and to the cell component of the product).
• In addition to preclinical study design considerations discussed in the Section titled “Toxicology studies” of this chapter, evaluation of the safety and activity of the cell/scaffold product will include appropriate controls, dose levels (scaffold seeded at various cell densities), and be of sufficient duration to allow for assessment of the biodegradation profile of the construct. The scaffold construct (synthetic or non-synthetic polymers) used in the pivotal preclinical studies will be identical to the intended clinical construct.

Preclinical study reports

The generation and submission in the IND of complete reports for each preclinical study conducted are important. Each complete study report will include, but is not limited to: 1) a prospectively designed protocol and listing of all protocol amendments; 2) a detailed description of the study design (e.g., animal species/model used, control and investigational products administered, dose levels, detailed procedures for product administration and collection of all study protocol parameters); 3) complete data sets for all parameters evaluated, including individual animal data and tabulated/summary data; and 4) analysis and interpretation of the results obtained.

E. Concluding Comments

This chapter outlines the major CMC and preclinical considerations to support the submission of an IND for an early-phase clinical trial using an investigational CGT product intended to treat cardiac disease. A primary goal of the resulting data is to sufficiently characterize the potential risks of the CGT product to inform the design of a clinical protocol that reasonably protects subject safety. In order to meet this goal, the source materials, manufacturing process, and final product need to be sufficiently characterized to provide an adequate assurance of safety. Additionally, data from relevant preclinical studies needs to be submitted in the regulatory submission with sufficient detail to allow for an independent review by CBER. This chapter represents an introduction to the regulation of the CGT products; however, it does not cover all aspects in the regulations and guidances. In addition, the development of novel cardiac CGT products can raise scientific and regulatory issues that may not be discussed in this chapter or in existing FDA regulations or guidances. CBER is cognizant of this scenario; thus the oversight of investigational CGT products includes enough flexibility to regulate this evolving field (20). Although FDA guidance documents are a good source of general information, for application-specific feedback, product developers and clinical investigators are encouraged to communicate with CBER early in product development, to enable timely progression from bench to bedside.

 

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