Preclinical Drug Development for a Gene Therapy Product: No Cookie Cutter Route


Follow these critical steps on your path to IND application.

By Philip J. Kuehl, PhD, Senior Scientist and Director of Scientific Core Laboratories, Lovelace Biomedical and Jacob D. McDonald , PhD, Senior Scientist and Chief Scientific Officer, Lovelace Biomedical

Cell and gene therapy products are reviewed and approved through the Center for Biologics Evaluation and Research (CBER) at the Food and Drug Administration (FDA). As of March 2019, there were 17 approved cellular and gene therapy products.1 With FDA’s trio of gene therapy approvals in 2017, the innovation floodgates opened. Pharmaceutical and biotechnology companies are seeking to capitalize on a revolutionary therapeutic approach that was once deemed too risky. With promising clinical results from early gene therapies, venture capital money is finally freeing up to enable this exciting next generation of medicine.

However, even the most well-funded gene therapy programs are exceedingly complex to navigate through preclinical development. While CBER has a specific set of guidances for cellular and gene therapies,2 each program is unique. These safety and efficacy tests, which are required before drugs can be studied in humans, are perhaps the most perilous stage of the entire development process. Drug companies must design studies that demonstrate, with sound data, that the therapy is safe and effective before regulators deem it suitable for dosing in patients.

The stakes couldn’t be higher. Depending on the outcome of preclinical safety and biodistribution studies, investigational gene therapies will either move to the final stage of FDA review or they will fizzle, sending scientists back to square one. The value of recent mergers and acquisitions clearly shows evidence of the value of the success of these studies and the development of cell and gene therapies.

Build an Expert Team

Building a team with a proven track record for designing and conducting safety and biodistribution studies on investigational gene therapies is the best recipe for successful completion of these studies. A poorly designed program could result in lost revenue, inefficient use of animals in research, generation of data that is not required, and worst of all, lengthening the time constant to submission of the investigational new drug (IND). This team should include scientific experts within the target disease pharmacology, dose formulation (vector and payload), toxicology, and regulatory sciences. Together, an experienced team with this composition can and will understand the nuances of designing and implementing a preclinical program supporting an IND submission that answers all regulatory questions.

As with any development team, the more specific experience the team has (in this case, approved IND programs to which they have contributed), the more drug sponsors can take advantage of previous experience through preclinical studies for eventual regulatory approval. One thing is clear: There is no cookie cutter path for gene therapy products. In an emerging therapeutic space, every step requires a customized and highly informed approach. While all programs should engage and interact with the regulatory authorities early and often, this is especially true for cell and gene therapies!

Detailed below are several critical steps within cell and gene therapy and some important considerations that any development team should take into account.

Select a Vector

How will the corrective cell and gene therapy product at the core of a gene therapy be delivered into patients’ cells? Typically, the answer is through a virus. Adeno associated viruses (AAV), adenoviruses, and lentiviruses are most commonly used as vectors for drug delivery. This choice hinges on therapeutic approach: AAV vectors provide long-term gene expression and naturally occurring sub-types (serotypes), which allows for some target-tissue specificity. Adenovirus vectors may be optimal when short-term gene expression is the goal. And lentiviruses are most often used to transfer genetic material to patient cells in culture (bone marrow or blood cells) that are subsequently injected back into the patient as therapy to treat immune deficiencies or sickle cell anemia.

Other modes of gene delivery include liposomes (lipid particles) and nanoparticles that can be engineered to target-specific cell types for delivery. The advantage with these is that they do not produce immune responses that are associated with viral vector use. The disadvantage is that often the composition of each of these must be specifically refined for each target disease and payload and, depending on the composition, may require and evaluation of immunogenicity.

Route of Administration

The route of drug administration depends on the target organ or tissue where the defective gene is expressed. For example, with ocular, joint, or brain-directed therapies, the vector is delivered directly to the eye, joint, or brain or spinal fluid. In the case of Pompe disease, which damages muscle and nerve cells throughout the body, the vector is given either intravenously or into the diaphragm or skeletal muscle. For cardiac therapies, the drug may be administered via catheter to the coronary vessels, or directly applied to the surface of the heart. For Alpha-1 antitrypsin deficiency, a genetic disorder that leads to lung and liver problems, the vector is administered intravenously or into the pleural space between ribs and the lung.

As for the timing of the therapy, this too is an important consideration for preclinical studies. With inherited diseases, it’s optimal to deliver the medicine to the fetus or infant as soon as possible after diagnosis. However, with few exceptions, FDA requires testing in adults or children at least 12 years old before allowing delivery to babies or small children.

Evaluate Biodistribution

Except for instances in which the vector is administered to confined spaces such as the eye or joint, experience has shown that gene therapies will distribute to off-target sites. However, this unwanted effect can be diminished by incorporating “promoters” within the vector to limit or control gene expression. Some unique promoters have sensitivity to light or oxygen tension to control gene expression. With Pompe disease, a desmin-specific promotor limits expression to a limited tissue set, including muscle, although the AAV vector distributes through the blood to most of the body’s tissues. Typically, biodistribution is measured with a quantitative polymerase chain reaction assay or with some type of immunohistochemistry end point. While the baseline equipment/technology/expertise for these methods are established, each one must be specifically developed and refined for each program. This includes defining the species and tissues for analysis. Early in the development program, a wide range of target tissues are evaluated and the list is reduced as the program advances. The program team should expect that a biodistribution assay will require validation to support the good laboratory practice (GLP) toxicology studies for regulatory submissions.

Select Species for Study

Choosing a species for preclinical testing of gene therapies is one of the most challenging decisions of study design. That’s because most gene therapies seek to treat rare diseases, which can be difficult or impossible to replicate in an animal. Some models will have a naturally occurring mutation, while others are genetically modified (as seen in the GAA knockout model of Pompe disease or Sandoff mouse model of Tay-Sachs disease). Animal models may be developed chemically, as with mono-iodoacetate-induced osteoarthritis, or through physical means, as with cardiac failure in pigs induced by vascular occlusion or electrical pacing. In addition, the sponsor may conduct in vitro studies to demonstrate to FDA that a given therapy will be taken up by cells of a chosen species in a manner similar to uptake in human cells, or that the receptors being targeted in human cells are also present in the animal species.

There are often many layers of complication influencing the drug sponsor’s decision around which animal models to use. This is based in part on the fact that you are delivering a human gene to an animal. And an animal may or may not respond to the human protein in the same way it would to a protein from its own species. That is one reason why a drug development team may decide to evaluate its therapy in two species, such as mouse and non-human primate.

The rationale for the species used must be justified in the pre-IND package and in the IND—and this is the reasoning behind the need for an experienced drug development team that leverage past experience from many other IND submissions. For example, while non-human primates can serve as a disease model for certain conditions, many primates naturally have some level of neutralizing antibodies to the vector that’s used to deliver the gene therapy—which means the animal would show no response to a gene therapy. For this reason, all non-human primates must be prescreened.

Additionally, when selecting the research facilities (often a contract research organization) to perform these studies, it’s critical to evaluate their level of veterinary talent. Many of the animal models and delivery methods require research veterinarians to conduct the surgical modes of delivery and the endpoint analysis. When measuring disease and response to treatment in an animal model, the team must be able to distinguish between the disease itself and the toxicological effects of the treatment, which requires skill and experience. In some cases, scientists evaluate efficacy and safety at the same time and in the same model.

Craft the Study Design

Many factors are taken into account when designing the preclinical study/studies, from number of dose groups, number of animals per dose group, types of controls, and number of endpoint-sampling time points. Unless a genetic disease occurs in only one sex, both sexes are included in safety and biodistribution studies. In most cases, at least two vector doses are used. Multiple sampling time points are included, beginning at the point when vector expression is to peak (usually 7 to 14 days) and extending for several months to one year.

Set the Critical Endpoints

Common endpoints for a gene therapy study include: body weight, clinical signs, hematology, serum chemistry, vector biodistribution (as evaluated by polymerase chain reaction), gene expression in target tissue and in tissues having a pre-specified large concentration of vector capsid (the shell of the virus), neutralizing antibodies in serum to capsid protein and transgene, immune responses (T cell-mediated to capsid protein and expressed protein), and histopathology (and immunohistochemistry for microscopic evaluation of gene expression). Other endpoints may be included, depending on the disease. Another key point to note here: If the drug sponsor will be seeking regulatory approval in Europe, an additional step may be required to evaluate vector concentration in bodily fluids and excreta to determine shedding.

Get Ready for IND

After preclinical results are evaluated using the latest bioanalytical tools and reporting, it’s time to complete the IND application, showing the strong data that indicates your drug is ready for testing in patients. All preclinical safety studies for gene therapies are conducted under GLP guidelines. An audited final report with summary data, statistics, and appendices containing contributing scientist reports and individual animal data is submitted to the sponsor for inclusion in the IND package.


  1. Approved Cellular and Gene Therapy Products. U.S. Food and Drug Administration. Published March 29, 2019. Accessed March 31, 2020.
  2. Cellular & Gene Therapy Guidances. U.S. Food and Drug Administration. Published February 14, 2020. Accessed March 31, 2020.

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