Gene Therapy Today: Notes from PPMD’s Duchenne Gene Therapy Forum

Gene therapy.

Those two words have been tossed around for some time, starting in the late 1990s when several gene therapy trials started, but ended with fatal results. Now, when the field has learned a lot from those initial results and with optimized technologies, we come back to gene therapy as a viable option for genetic diseases. This renewed interest has gone hand-in-hand with the focus on rare diseases. As more is learned about the genetic causes of rare diseases, the most effective strategy for a cure would be to impact the genetic cause itself. 

There has been much interest in gene therapy for Duchenne as of late, and it seemed like a perfect time to gather the community to discuss the status of gene therapy development, the remaining challenges and ways the community can move forward together. PPMD held a Gene Therapy Forum on February 22nd at the Mayflower Hotel in DC, with FDA, industry, and families and patients at the table.

FDA Kicks Off Gene Therapy Forum

Dr. Celia Witten of the Office of Cellular, Tissues and Gene Therapy (OCTGT) of the Center for Biologics Evaluation and Review (CBER), FDA, started the meeting off with an overview of her office, what it does, and some of the resources available to support development in gene therapy (click here to download Dr. Witten's slides). Her office provides regulatory review for human tissue/tissue products for transplantation, cells, and gene therapy. Additionally OCTGT provides regulatory policy and guidance development, manages international activities, does outreach, and oversees publications and mission-relevant research. OCTGT has experienced growth in the last few years as evidenced by the increased number of Investigational Device Exemption (IDE) and Investigational New Drug (IND) applications with most of the growth coming from gene therapy as compared to cell or tissue based therapies. The office offers many resources, such as a Learn Webinar Series, and various guidances.

Click here to download Dr. Witten's slides (PDF)

She ended her presentation with comments about several of the expedited programs that the FDA offers. In particular, OCTGT has received 43 requests for breakthrough designation, 24 of them from oncology and the rest from non oncology. Of the applications received, 11 were granted breakthrough status, 7 of them from oncology and 4 from non-oncology. She encouraged attendees to use the resources that her office provides.

The State of Gene Therapy Development

The second portion of the meeting focused on an update on the current state of gene therapy development in Duchenne and focused on four topics:

  1. Entry into the cell via Adeno-associated virus (AAV)
  2. How to Fit the Dystrophin Gene into the AAV
  3. Transfer of Genes other than the dystrophin gene, as potential therapeutics for Duchenne
  4. Experience of Gene Therapy in Spinal Muscular Atrophy (SMA)

Panelists at PPMD's Gene Therapy Forum

The Adeno-Associated Virus – A Way into the Cell

The first talk covered the Adeno-associated virus which is how the dystrophin gene will get into the body in gene therapy. It is kind of like a container, and just like a virus that makes you sick, can enter a cell and start replicating what DNA is inside the container. If the dystrophin gene is put into a cell nucleus via AAV, it will start making dystrophin.

The first presenter, Dr. Jim Wilson from University of Pennsylvania, has been involved with AAV and gene therapy for years. He talked about how gene therapy was first tried with an Adeno virus which is very inflammatory so a strong immune response was evoked. Although the gene produced a lot of protein, the effect was lost over time because of the strong immune response. So attention turned to the Adeno-associated virus, a virus that resides within us in a latent form, causes less inflammation, and in studies has shown the potential for stability of expression. In theory, this vector can deliver its “payload” (genetic instructions it is carrying) and remain stable for a number of years.  AAV2, one of the earlier vectors used, was shown to be able to get into the cell and not invoke an immune response, showing expression for over 11 years.  Over time, Dr. Wilson has worked on developing better vectors such as AAV8 and AAV9, that are able to invoke even less of an immune response and target certain tissues, such as heart and muscle tissues. The vector for Glybera, a gene therapy product approved in Europe for Lipoprotein Lipase Deficiency, uses an AAV vector that was developed in Dr. Wilson’s lab.

Specific to Duchenne, there are several challenges that have to be overcome. The first is delivery to muscle as that is where the dystrophin is needed. AAV9 has shown a remarkable ability to cross the blood barrier into muscle in monkey and dog, and research is continuing.  A second specific challenge for Duchenne is safety, as there still is the possibility for an immune response to the vector. As discussed above, AAV has been chosen as it evokes the smallest immune response but this is still a challenge as Duchenne is an inflammatory disease to start with and even the slightest perturbation could cause a response.  Using an immune suppressor, such as steroids, is being discussed as a way around this. And last, vector scale up and supply was discussed. In systemic delivery, vector must be delivered to the whole body ideally, which equals about 20 liters per patient. Consistent, repeatable production processes must be developed to ensure that enough high quality material can be manufactured to meet the needs of the patient population. All these issues are being worked on and several companies are moving ahead with preparing to move into the first small human clinical trials in the next couple of years (including Bamboo & Solid).

How to Fit the Dystrophin Gene inside the AAV

Dongshen Duan of the University of Missouri discussed work that has been done to fit the large dystrophin gene into the AAV. The AAV has a carrying capacity of 5 kilobases (kb). However the dystrophin gene is much larger than 5 kilobases and thus can’t fit inside the AAV. To overcome this challenge, a smaller dystrophin gene needed to be created that was still functional. Mirroring a patient that had a deletion of exons 17-48, a minimized dystrophin of 6 kb was made. But this still wasn’t small enough.

Dr. Duan, and many other researchers, have worked diligently to find ways to modify this dystrophin, finding out which portion of the gene could be modified without losing function.  They developed several models of a functional miniaturized dystrophin of 5 kb (or even less). This micro dystrophin has been injected systemically into dogs and mice and good gene transfer has been seen in both species in muscle and heart.  In mice, the histopathology of Duchenne has been dramatically reduced and muscle strength has increased.  In the dogs, efficient gene transfer was seen in major and minor muscle groups, including the heart, and reduced fibrosis and scarring was observed.  A single IV injection of of this gene therapy, with the use of immunosuppression for five weeks, resulted in a lasting effect for nine months. These promising results show that a miniaturized truncated dystrophin gene can be effective at producing functional dystrophin in gene therapy and this concept is being taken forward in development.

Could Other Genes be Transferred to Achieve a Therapeutic Effect?

Dr. Kevin Flanigan of Nationwide Children’s Hospital discussed two alternative genes to dystrophin that could be delivered that may have benefit for Duchenne patients. These wouldn’t be essentially correcting the gene defect itself but causing expression of another helpful protein – in one case GALGT2, and causing exon skipping in another case. 

GALGT2 is found in the sarco-glycan complex and is involved in utrophin upregulation, which is helpful for Duchenne as it prevents loss of strength. Immune issues are minimized in this strategy as GALGT2 is found naturally in our bodies so our bodies have already “seen” it, so it is not new. The same immune issues that stem from delivery via the AAV are present here though and, as discussed above, researchers are working to overcome this safety challenge. A human clinical study is being planned using AAV-GALGT2 administered as an IM injection. Additionally, GALGT2 shows broad therapeutic application to other muscular dystrophies so there is considerable reason to move forward.

A second alternative gene therapy is AAV mediated exon skipping, using U7snRNA, meaning that the exon skipping instructions are contained in the U7snRNA and they are carried into the cell via AAV. This strategy has been successfully explored in a mouse model where exon 2 is duplicated, also developed by Dr. Flanigan. The U7snRNA is designed so that only one of the duplicated exons is skipped, essentially taking way the defect, so normal dystrophin can be produced. Dr. Flanigan has shown in his dystrophic mouse model with an exon 2 duplication that treatment with the AAV-U7snRNA creates a functional dystrophin protein in the right place at the cell membrane. In addition to providing a useful tool for the evaluation of potential exon duplication skipping strategies, the new mouse model provides a tool to study a novel feature of dystrophin discovered in the Flanigan lab, called an internal ribosomal entry site (IRES), found within exon 5.  When this site is turned on, truncated but functional dystrophin is made. Dr. Flanigan is exploring IRES as a potential strategy for mutation in the first few exons of the dystrophin gene. We are hopeful that these alternative gene transfer strategies prove useful to Duchenne patients.


What Can We Learn from Other Gene Therapy Clinical Trials?

Dr. Jerry Mendell, of Nationwide Children’s Hospital, presented results from an ongoing gene therapy trial in Spinal Muscular Atrophy (SMA). His trial focused on SMA type 1, which results from a mutation in the SMN gene and in which only 8% of patients live beyond 20 months. In this Phase 1 trial, 15 patients were enrolled, and received an IV low dose or high dose. All patients were on prednisone starting the day before treatment so immune responses were minimized. All patients were also pre-screened for antibodies to SMN, and if these levels were too high, they were excluded from the trial. There has not been a single death in this trial of infants, and there seems to be a dose response as those receiving the higher dose had better clinical outcomes. Two patients had elevated liver enzymes which resolved with prednisone suppression. Dr. Mendelll emphasized that the age of the patient seems to influence the outcome of the therapy. This is important as we consider how we go about gene therapy in Duchenne and also, supports efforts to include Duchenne as a routine part of the Newborn Screening Panel – efforts PPMD feels passionately about.  In Dr. Mendell’s earlier Duchenne gene therapy trials, a strong immune response was seen, most likely from revertant fibers containing dystrophin. He encouraged checking gene therapy candidates for pre-existing immunity to dystrophin prior to gene transfer.

The Challenges Ahead

The third part of the meeting was a panel discussion on the challenges for gene therapy in Duchenne.  The panel included included Dr. Carrie Miceli, Dr. Flanigan, Dr. Mendell, and three CBER OCTGT representatives; Dr. Mercedes Sarabian, Dr.  Lai Xu, and Dr. Denise Gavin.  

Research challenges discussed included:

  • Overcoming the immune response via immune suppression
  • The opportunity of delivery into younger children as their body mass is less
  • The need to balance target age with endpoint performance as it may be harder to see improvement in younger children
  • The issue of producing at the scale that is needed 

Pre-clinical challenges discussed included:

  •  The importance of robust and reproducible pre-clinical data
  •  Which species of animal do you use for which test
  •  Dose and immune response extrapolation from animal to human
  •  The quality of definitive safety studies.

Clinical challenges discussed included:

  • Appropriate design of the early phase studies
  • Special considerations needed to go into a pediatric population with the benefit /risk profile of Duchenne
  • Pros and cons of including a control arm

Manufacturing challenges discussed included:

  • Product characterization issues as much of the early work is being done in labs and then transferred to industry 

While no concrete answers were given for these challenges, some ideas came up such as the use of reference materials, data-sharing, standardization, and the value of sharing both negative and positive results.  

Finding a Path Forward

The last panel of the day focused on ways the community could work together and create a path forward.  It seemed that several issues rose to the top as those to be focused on.  There was much discussion on what would be the appropriate endpoints for a gene therapy trial and how could ICE (intermediate clinical endpoints) be applied.  Endpoints need to be re-examined and especially in the context of gene therapy and potentially going into younger children.

What would be appropriate clinical endpoints? Patients have an important role in developing endpoints as we move forward as they are the ones to notice the more subtle things on a daily basis that make a difference for the patient. Novel ways of designing trials to minimize the number of patients needed was also discussed, although this would require the collaboration with companies, regulators, and patients.  With gene therapy trials still so new, there is much for families and patients to learn to make an informed choice as to whether gene therapy is right for them. Because not all gene therapies are the same, at a minimum our community needs more education as to the kinds of questions to ask before entering a trial.


Much has been learned from past experiences that can be used to develop a tool for parents as they consider entry into a gene therapy trial. A specific issue for the community to grapple with is that of the impact of exposure to a virus from a gene therapy trial. Once you are exposed, does that mean you can not be eligible for another trial that uses a viral vector? Or even another dose? Are there differences in different virus serotypes that could be a workaround here?  These are just some of the questions that need to be laid out as gene therapy gets closer to the clinic. There may be no one answer. As with every clinical trial, participation is a personal and family decision.

In summary, gene therapy is upon us, great strides have been made, and yet there is much to learn still. The data is promising but we have not yet entered humans so what we have now probably isn’t what we’ll end up with. We are still learning pre-clinically how to proceed as safely as possible which is why, as with any drug development program, progress for now will seem methodical and cautious. 

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Comment by Shelly on March 9, 2016 at 7:45pm
I am glad to read the success story of UCLA RESEARCHERS. Their work is highly appreciable. They corrected the stem cells with the help of CRISPR and then developed ips cells into cardiac and skeleton cells and transplanted them in animal models. Surprisingly, dystrophin was produced in the transplanted cells. It's something indicating the scientists are close to end duchenne. PPMD's work will certainly pay one day. May the day comes quick.
Thanks to PPMD.
Comment by Shelly on March 9, 2016 at 7:21pm
Many thanks Abby!!!
Fingers crossed! May something can be done for the boys before time slips out of our hands.
Comment by Abby Bronson on March 9, 2016 at 2:36pm

Hi Shelly - Good question. CRISPR was not discussed in the gene therapy forum because, although it is related, it isn't the same as gene therapy.  Gene therapy for Duchenne seeks to deliver a copy of the dystrophin gene, in hopes that it produces full length dystrophin.  CRISPR uses a system of enzymes that cut DNA and single strand RNA that guides the enzymes to the right place within the DNA to cut. CRISPR  will be used to essentially cut the DNA so that it makes an in frame deletion, and a truncated but functional dystrophin will be made. This is similar to what exon skipping is doing, but getting there in a different way.  In both gene therapy and CRISPR, AAV is used to deliver either the gene or the CRISPR/Cas9 system to the muscle cells.  What we learn in gene therapy about using AAV, we can apply to CRISPR delivery.  Here are two links that you may find helpful.

Comment by Shelly on March 8, 2016 at 7:38pm
CRISPR is not discussed in the above gene therapy forum. Is it something different than AAV gene therapy. What is the difference between two of these therapies?

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