#### Demonstration of full spectrum animal model from single mutations is optimistic for gene therapy Three different Midwestern NF centers with mini swine facilities ([Iowa](https://www.ncbi.nlm.nih.gov/pubmed/29925695), [Minnesota](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168575/) and [Wisconsin](https://news.wisc.edu/nf1/)) have claimed full-spectrum reproduction of NF1 complications in miniswine, with a single gene mutation. To the extent that a single mutation can reliably produce an NF1 complication, gene therapy becomes attractive. Using mini swine can rapidly accelerate the testing cycle for either drug or gene therapy. We might also be able to speed up the testing cycle by another order of magnitude by, for example, using NF1 mini swine [organoids ](https://www.drugtargetreview.com/article/48244/what-are-the-pros-and-cons-of-using-organoids/)(or just human tumor organoids). Recently conditions for gene therapy have become more promising due to [CRISPR](https://www.sciencemag.org/news/2019/08/forget-single-genes-crispr-now-cuts-and-splices-whole-chromosomes). #### The only known attempted NF1 gene expression therapy failed due to incorrect hypothesis about NF1 +/- availability in tumor microenvironment I only know of one attempted gene expression therapy for NF1, by David Segal at UCSD. He [attempted an approach, with a negative outcome, 5 years ago](https://apps.dtic.mil/dtic/tr/fulltext/u2/a609751.pdf). In email communication to me, Prof. Segal described the reasons for the negative outcome as follows: > Our approach tried to take advantage of the natural biology of TALE-type artificial transcription factors. In nature, TALE proteins are already gene activator proteins, and they are produced in bacteria which transmit the TALE proteins to their host. The plan was to engineer TALEs that could turn up the expression of the good copy of NF1 (most people with NF1 inherit one good copy and one mutant copy). Bacteria producing the engineered TALEs would then be injected into the fibromas, where they would remain to pump out the TALE until the tumor went away. They they would be exposed to, and cleared by, the host immune system. > The first problem was that the natural bacteria of the TALEs does not grown in humans, so we had to use a different strain of bacteria. That already raised concerns by some in our lab safety. > The second problem was the realization that what actually happens in the fibromas is that the good copy of NF1 is lost, leaving only the mutant copy. That means there was no good copy to increase the expression of. So our plan then switched to targeting the therapy to the cells in the microenvironment of the fibroma. There was what appeared to be pretty convincing evidence in the literature (mostly Science and Cell papers) that the NF1 expression in the microenvironment was absolutely critical for the fibroma to form and persist. That was what we wrote into the grant, and the grant was reviewed and approved. However, as we talked with more and more scientists, they started to cast doubt about the necessity of NF1+/- status in the microenvironment, now that there have been a lot more studies. > So yes we had a partial success with the methodology, and yes it would in principle work with any type of NF1 mutation. > However, I think the approach we were taking had no future. #### A recent startup focuses on enhancing normal tissue expression of neurofibromin 1 One startup, http://www.infixionbio.com/our-approach/ focussess on enhancing the ability of normal tissue to produce neurofibromin 1. To summarize their website: * NF1 mutations occur on one of 2 alleles in the NF1 gene (autosomal dominant), so there is always a health copy present. That is, there are no observed individuals who are NF1 -/- in non-tumor tissue. * It has been shown that the lack of a healthy amount of wild-type neurofibromin 1, not the presence of mutated neurofibromin 1, causes the complications seen in NF1 * The approach is to amplify the healthy allele: "Utilizing cutting-edge tools and techniques in gene regulation, gene transcription, protein life-cycle management, pluripotent stem cells, and high-throughput screening, the Infixion Bioscience research focus is to develop a safe and mild drug that enhances the NF1-affected person?s natural ability to both generate and conserve normal Neurofibromin protein produced by the unmutated, or wild-type, allele. This increase in the amount of Neurofibromin has the potential to mitigate many, if not ALL, downstream NF1 symptoms that result from [haploinsuffiency](http://jem.rupress.org/content/191/1/181) of Neurofibromin. Similar to insulin treatments for diabetes, a drug that increases NF1 protein on an ongoing basis has the potential to deliver a functional cure for NF1." So, their approach is to stimulate additional production of neufibromin 1 by the wild-type allele which, they say, is always present. However, note that Dr. Segal's experience was that the tumor microevironment is NF1-/-, which may limit the effectiveness of this approach, in case the normal tissue expressed neurofibromin 1 cannot find it's way into tumor cells. (Which may be by way of definition of tumor cells.) #### The case for gene mimetic therapy The wild-type [neurofibromin 1 protein](https://en.wikipedia.org/wiki/Neurofibromin_1) is a GTPase-activating protein that negatively regulates RAS/MAPK pathway activity by accelerating the hydrolysis of Ras-bound GTP. As a Hackathon deliverable, can we produce a summary of the receptor sites and signalling pathways acted on by neurofibromin 1? Then we have two mimetic approaches: * Identify chemical structures of known drugs which can bind to those receptor sites and act similarly to accelerate the hydrolysis of Ras-bound GTP * Synthesize neurofibromin 1 directly by way of some organic chemical reaction, or by cloning neurofibromin 1 by some standard protein replication process Then we need to assess if either a chemical analog or synthesized neurofibromin 1 is deliverable into cells by some method (injection, ingestion, topical cream), and once delivered will it act the same way as wild-type neurofibromin 1 produced by the body. Direct delivery to tumor cells may be necessary to overcome the naturally NF-/- status of the tumor microenvironment, as the presence of a tumor may be defined by the tumor microenvironment's ability to maintain NF-/- status. #### How does the tumor maintain NF-/- status? What are the mechanisms by which a tumor excludes NF+/- cells in favor of NF-/- cells? Can this mechanism be defeated? If so, would defeating NF+/- rejection be a therapy, or is it a necessary antecedent to delivery of neurofibromin 1 or a mimetic?