Meeting of the Minds

Most scientific meetings strive to inform the audience on the state of the field by presenting data in a consolidated manner, maximizing speaker time often at the expense of discussion time. Separating clinical and scientific sessions is common. This year, when planning our Investigator Meeting in Boston on May 21 – 24, Monica and Randy had the foresight to take a different approach.

They decided what we really needed was a “Meeting of the Minds” where our basic scientists and expert Rett clinicians could update one another on the current state of the field while keeping the focus on the key questions remaining to be addressed and the main obstacles to success. This potentially risky and unusual format focusing on challenges and difficulties, designed to foster active discussion and encourage brain-storming and problem-solving from new angles, was not only energizing and provocative, but effective. New approaches, out of the box thinking, and the seeds of new collaborations were sewn.

The social aspects of the meeting helped to strengthen existing relationships and foster new ones. The investigators appreciated the opportunity to keep an eye on the ultimate goal by meeting several local children with Rett and learning about their lives. It was heartwarming to see the families acknowledge and appreciate the efforts of the Investigators to get to the bottom of this disorder.

The meeting started with the basic function of MECP2 and gene therapy approaches in development, followed by key tools and model systems in studying MECP2, MECP2 reactivation strategies and progress, and concluded with clinical research learnings and outcome measures to assess therapeutics.

I came out of this meeting feeling extremely excited and optimistic about where things are heading, but also aware that challenges still lie ahead. In summary, the mutations that cause Rett Syndrome broadly impair brain function through complex mechanisms that are not completely understood. It is not surprising that addressing the root cause of Rett Syndrome by restoring MeCP2 protein in brain cells is profoundly more effective than therapeutics that target disruptions in one of the numerous downstream pathways. Because the mouse and human MECP2 genes are not identical, it will be necessary to optimize many of the newer biologic therapeutics (e.g., RNA modifications, MECP2 reactivation) in the human cell lines with stable X-inactivation that are currently being generated. While there is optimism that initial gene therapy will provide significant benefit, additional next generation gene therapy programs will continue to be pursued in parallel. A shared goal for these cure-focused programs is to obtain uniform delivery to all brain cells and effectively optimize MeCP2 protein levels in each cell.

For anyone who wants to delve further into the research, below is more detail on what was presented in each session and some of the outstanding questions that will need to be addressed.

MECP2 Function

We know MECP2 regulates the expression of hundreds of genes and that behavioral changes in mouse models of Rett are difficult to assess in a meaningful way when evaluating potential therapeutics. The severity of MECP2 mutations in humans correlates well with the survival time of male Rett mice, suggesting that mouse survival may be a good marker for the potential success of therapies.

A lively discussion ensued regarding the minimum level of MeCP2 protein required to improve cellular function, and what percentage of cells must attain this level to provide a therapeutic benefit. It was noted that survival in the mouse Rett model is meaningfully prolonged when MeCP2 protein levels are greater than 5-10% of normal in all cells or when normal levels of protein are achieved in 10-40% of cells.

Genetic Approaches: DNA and RNA

The most thought-provoking session at the meeting was gene therapy. A lively discussion acknowledged that gene therapy is a new therapeutic approach and that we are all pioneers. Avexis’ Brian Kaspar indicated that all of the data points to a well-tolerated, non-toxic, gene therapy product, and presented efficacy results for AVXS-201 (heading into clinical trials early next year) that are profoundly better than any drug previously tested in mice.

Subsequent presentations highlighted the risks associated with gene therapy as well as those unique for Rett Syndrome. However, we have confidence that FDA has sufficient experience with the gene therapy field to assure the study is as safe as possible for study subjects. Key unknowns will remain until the first clinical trial: will the MECP2 gene be delivered to a sufficient number of brain cells, and will the delivered gene produce an optimal amount of protein in each cell? We will not be able to reliably predict the magnitude of efficacy expected in this first trial. With this in mind, we will continue research in parallel focused on enhancing delivery to the brain and regulating the amount of MeCP2 protein produced.

In a new and up-coming area of research, RNA modification, scientists are working to hijack existing cellular machinery to correct mutations at the level of RNA and thereby restore normal MeCP2 protein levels in each cell. Although considerably less advanced than gene therapy, these approaches offer advantages for regulating the level of MeCP2 protein in each cell. The RNA editing approach being pursued by the Mandel lab demonstrated the highest correction rate reported to date at a whopping 72%. RNA transplicing is an alternative approach that has the potential to correct over 97% of all MECP2 mutations with a single therapeutic. Current efforts are focused on optimizing the efficiency for modifying RNA and rapidly advancing development of these therapeutics toward clinical trials.

Induced Pluripotent Stem Cells

A key tool for developing many of the newer Rett therapeutics will be human induced pluripotent stem cells, hiPSCs. hiPSCs can be used to generate any other type of cell, allowing scientists to generate brain cells from Rett Syndrome patients. Scientists from academia and industry reported on progress in utilizing hiPSCs to discover and develop novel therapeutics for Rett Syndrome. Another tool being used to study Rett Syndrome is to generate organoids, or a “brain in a dish”, from hiPSCs. Organoids grow into more complex cell types and layers and provide informative ways to assess cell signaling.

A unique issue in studying Rett Syndrome hiPSCs is that X inactivation, or silencing of one of the two X chromosomes in female cells, can degrade in these cell lines and we still don’t know how to fully control this consistently. It is important for the field to understand and control X-inactivation in hiPSCs not only to facilitate therapeutic development, but to explore the potential for exploiting underlying mechanisms to reactivate MECP2 on the silent X chromosome.

MECP2 Reactivation

A key regulator of X inactivation is Xist, a non-coding RNA that coats the chromosome leading to its silencing. But is loss of Xist enough to turn on gene expression or do we need an additional activation step? Loss of Xist appears to have no negative effects in mice and can result in 2-5% reactivation of MECP2. A 4-10% increase in MeCP2 protein levels provides a 25% increase in mouse survival.

Alternative approaches to activate MECP2 employing CRISPR/dCas9 or Zinc Finger guides are also being pursued. Recent experiments have shown that changing the state of the gene from a silenced signature to an active signature is enough to turn a gene on from the active X chromosome and conditions are being optimized to turn on MECP2 from the silenced chromosome.

Some key questions that remain are: Can this method be used in neurons and are the effects long or short-term? Will there be an immune response to the novel proteins administered to activate the gene? Can we use existing drugs to enhance the efficiency of MECP2 expression?

Clinical Research

The Natural History Study has enrolled almost 2,000 patients to date and the findings from this observational study have resulted in 30 publications with several pending or in preparation. Current efforts are focused on making these data more accessible to families, supporting industry and clinical trials in Rett Syndrome, and developing improved clinical outcome measures. Additional goals include standardizing clinical care and identifying biomarkers.

Neurodevelopmental disorders are historically difficult to study and many successful therapies in mouse models fail to translate to efficacy in humans. It was suggested that paradigm shifts at FDA may be required with a focus to study the whole disease rather than specific behaviors. Physicians, advocacy groups, and industry are urged to engage FDA to develop improved assessments for efficacy in clinical trials. Can assessments in human cells improve the likelihood of efficacy in clinical trials for Rett Syndrome?

Novel Outcome Measures

Measuring change in disease symptomology is key to finding and demonstrating successful therapies. Rett specific severity scales that are better able to document meaningful improvements for individuals with Rett Syndrome and their families are clearly needed. A number of physiologic measures (brain waves, breathing patterns) are altered in Rett patients and could become objective biomarkers or outcome measures.

In addition to typical outcome measures performed during a clinic visit, obtaining unbiased, objective data of a patient’s status over a continuous period of time is emerging as an area of active development. The potential to employ biosensors and video recordings in the home environment provides an opportunity for continuous measurement of health, function and quality of life in a real world setting. Additionally, cellular protein and metabolic signatures are being explored to define disrupted cellular networks and potential corrective therapeutics, and to identify biomarkers for use in clinical trials.