What's happening in the field of gene therapies for ataxias (Hayley McLouglin, PhD).

NAF 2023 Lecture Notes by Márcio Galvão (3/5/23)

Beverly Davidson, PhD

Hayley McLoughlin, PhD

https://www.youtube.com/watch?v=032LgFnBMyE

The Davidson laboratory is working on ASO gene therapies (see Ref. 1) for the ataxias SCA1 and SCA2. Studies are also underway to develop ASO therapy for the ATXN2 (ataxin-2) gene as a modifier of ALS (Amyotrophic Lateral Sclerosis). This is the SAME gene as the ataxia SCA2, although SCA2 is a very different disease from ALS (see Ref. 2). Gene therapy is also being investigated for Huntington's disease, which, like SCA1, SCA2, SCA3, and several other ataxias, is caused by abnormal polyglutamine (PolyQ) expansion.

The idea is that the genetic techniques being developed for these specific diseases can later be adapted for a variety of other neurological diseases.

Ref. 1 - https://www.ataxia.org/scasourceposts/snapshot-o-que-e-um-oligonucleotideo-antisenso-aso-aon/

Ref. 2 - https://pubmed.ncbi.nlm.nih.gov/27531668/

Some issues discussed by Dr. Beverly

1. What vectors can be used to deliver genetic drugs to specific cells?

An important aspect of ASO therapies is how to efficiently and safely deliver the drug (without serious side effects), reaching only the neurons that are "sick" (due to mutated genes) without affecting other "normal" or healthy cells. One option is to use AAV (Adeno Associated Virus) as viral vectors for drug delivery. Viruses have evolved over millions of years to penetrate human cells. Gene therapy takes advantage of this natural phenomenon. In this case, modified, harmless viruses are used as vectors, or carriers of the "genetic cargo" to be included in the affected cells. This is a proven safe method, which has been refined for 25 years and is already approved by the FDA for the treatment of several diseases.

2. How is a viral vector (rAAV) made?

Scientists remove the natural genetic load (DNA) of a virus (e.g., adenovirus) and replace it with the genetic material they wish to introduce into the patient's cell. Vectors can, for example, carry a healthy copy of the gene to replace the mutated copy in the patient's cell (= gene replacement), or use editing techniques such as CRISPR/Cas9 or RNA Interference (RNAi) to silence the mutant gene (= gene silencing), preventing it from synthesizing malformed proteins that can kill neurons, ultimately causing the symptoms of ataxia (neuronal loss, causing atrophy of the cerebellum and other nervous system structures).

3. How is the virus with the cargo introduced into the patient?

It can be by injection or infusion, depending on the disease that you want to cure with the therapy.

4. How do these rAAV vectors access target cells?

The vector naturally binds to receptors (receptor binding) found on the surface of cells. Genetic engineering is crucial here, as the goal is for the vectors to bind to the correct cells in the patient's brain, not to cells elsewhere in the body. After binding to the receptors on the correct cells, the viruses enter the cells in a compartment called the endosome (through a natural process called endocytosis).

Once inside the cell, viruses have naturally evolved to escape the endosome and migrate to the cell nucleus, where the genes they want to target are located. Once in the nucleus, the genetic code carried by the virus is converted into DNA (= transcription of the gene of interest occurs).

Dr. Davidson explained that in some cases, viruses already exist that can be used as vectors; in others, the viruses themselves are genetically engineered in the laboratory. The best technique depends on the desired therapy and the cell type targeted (e.g., liver cells or brain neurons, etc.), which varies depending on the disease being treated with gene therapy. For example, in the case of spinocerebellar ataxias (SCAs), the Davidson laboratory (and others) seek to optimize these viral vectors (using genetic engineering techniques) so that they bind specifically to Purkinje cells within the patient's cerebellum.

In addition to customizing the carrier virus, laboratories also customize the genetic material carried by the vector. In the case of spinocerebellar ataxias (SCAs), as already mentioned, one of the goals of therapy may be to *silencing* the gene that produces the malformed protein, preventing the gene from creating these defective proteins. It is the accumulation of these proteins in the nucleus of nerve cells that harms them (and causes ataxia). To silence mutated viruses (stop their "expressiveness"), a technique called RNA interference (RNAi) or editing of the mutant gene with CRISPR can be used, as we will see later.

5. The RNAi approach

RNA interference (RNAi) is a method of silencing dominant alleles (genes).

REVIEW: Genes contain messenger RNA (mRNA) that encodes proteins (for example, the ATXN1 gene encodes the ataxin1 protein, the ATXN3 gene encodes the ataxin3 protein, etc.). If the gene is mutated, the protein encoded by its messenger RNA is malformed and can disrupt various cellular functions (gene transcription, errors in gene expression, calcium homeostasis, etc.). This eventually leads to the death of the nerve cell (neuron). This is the problem that underlies spinocerebellar ataxias (SCAs)—the defective ataxin1 protein causes SCA1, the defective ataxin3 protein causes SCA3, etc.

RNA interference (RNAi) is a technique designed to interfere (as the name suggests) with messenger RNA, so that it no longer functions and synthesizes the bad protein. This way, without the defective protein, the problems are prevented at their root.

This approach has already been successfully tested in mouse models, producing significant improvements in SCA1 ataxia symptoms (Ref 3). Cellular functions previously compromised by the harmful protein improved, and symptoms, including motor performance, were also improved in the mice.

Ref 3. Megan Keizer, Brain 2015

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4802374/

The method's safety was also tested in primates, which have larger and more complex brains than mice, to assess safe dosages before administering the drug to human patients. These tests with non-human primates revealed a toxicity issue due to the vectors. After extensive study, the viral vectors were refined and tested again in mice. The results were positive. The improved motor performance of the mice reached almost the same level as that of normal mice (without ataxia). Now, this new RNA interference (RNAi) approach needs to be tested again in monkeys, and then discussions with the FDA are needed to begin human trials aimed at treating SCA1 ataxia.

6. CRISPR/Cas9

CRISPR/Cas9 is another method of somehow disabling the mutated gene, preventing it from producing defective proteins. In this case, the goal is to directly edit the gene (gene editing) rather than silencing it.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a large protein composed of small sections of bacterial DNA containing CAG nucleotide repeats (Ref. 4). Coupled with another component (Cas9), the CRIPSR protein is guided to the gene to be edited (for example, the ATXN2 gene, whose mutation causes SCA2) by a molecule called "guide RNA" (gRNA). This makes it possible to delete a specific section of DNA and remove (for example) the excessive CAG repeats of the mutant ATXN2 gene, thus ensuring that the encoded proteins are non-toxic, which is another way to prevent ataxia from developing in that individual. Because the CRISPR protein is so large, it needs to be packaged in two viral vectors. Each vector carries a component (Cas9 and gRNA). In tests in mice, the results were good. It is being verified that the levels of defective ataxin2 (in the case of SCA2) were in fact reduced by the CRISPR/Ca9 method.

Ref. 4

https://pt.wikipedia.org/wiki/CRISPR

7. Safety considerations

For these methods of treating ataxias (SCA1, SCA2, SCA3, and others) to be approved by the FDA for use in humans, they must be proven effective (= works) and safe (= does not cause harmful side effects). One problem with the CRISPR/Cas9 approach is that the protein comes from bacteria—it is not normally produced in the human body. Therefore, our immune system will attack this protein, as if the intended gene therapy were some kind of infection. So, how can we ensure safe delivery of CRISPR/Cas9?

One technique involves using viral vectors to introduce CRISPR into the cell, but keeping it "off" (silent) so as not to trigger immune responses, and turning CRISPR on only when needed. One method involves the patient taking (orally) a single dose of a drug containing a molecule (LMI070) that "activates" CRISPR, allowing it to be "assembled" correctly. This method is called "X-On"). In other words, the CRISPR gene-editing mechanism would be "drug-induced" at specific times, remaining off until activated, thus avoiding immune responses.

This "X-On" technique was tested in mice. First, the CRISPR was delivered "deactivated," and after a few weeks, the mice were given the drug LMI070 (an oral dose of a few milligrams), and the CRISPR was assembled correctly and became functional. The results were very encouraging.

8. Conclusions

Emerging gene therapies based on RNAi and CRISPR/Cas9 hold promise for the treatment of several neurological diseases, including ataxias. The Davidson laboratory's results for SCA1 and SCA2 are encouraging.

Dr. Davidson explained that much more testing is needed to determine the correct dosages for human patients, as well as methods for targeting the desired cells and avoiding toxicity, aiming for efficacy and safety, including safety after months of treatment, not just weeks or days. Using the "X-On" technique to activate CRISPR only when needed is important from a safety perspective.

According to Dr. Davidson, achieving results in humans with these ASO therapies today would still require a very high dose, so techniques still need to be optimized to allow the treatment to work with much lower doses, and therefore with much greater safety.

From what we know from mouse models, a 20% to 30% reduction in toxic protein levels is sufficient to improve ataxia symptoms. For human patients, the necessary reduction level is not yet known; this will only be determined through FDA-authorized clinical trials, where multiple doses of the drug can be tested.

Each of us inherits two copies (alleles) of each gene, one from our father and one from our mother. This also applies to the genes related to the ataxias SCA1, SCA2, SCA3, and so on. In mouse studies, it appears safe to silence some genes, preventing them from producing any proteins. However, in humans, the impact of silencing both copies of the gene is uncertain. For this reason, techniques are being developed that allow silencing only one of the alleles (the one with the mutation, inherited from either father or mother) while leaving the other normal allele intact. This, however, is complex. It is already known how to do it for Huntington's disease, and studies are underway to determine how to do it for the ataxia genes as well.

Once safety concerns and delivery mechanisms (viral vectors) have been optimized for use in humans, the techniques discussed here for Huntington's disease and the ataxias SCA1 and SCA3 are potentially adaptable to other types of "PolyQ" ataxias caused by abnormal trinucleotide repeats.

In closing, Dr. Davidson emphasized the importance of natural history studies in helping convince the FDA that therapies are effective and safe, as well as understanding which biomarkers can be used to monitor disease progression during trials.

[MG Note] Recently, the pharmaceutical company BIOGEN announced the suspension of its studies for the drug BIIB132, based on ASO therapy for ataxia SCA3. However, the drug VO659, also based on ASO therapy from Vico Therapeutics, for SCA1, SCA3, and Huntington's disease, continues in the NAF pipeline. Progress can be monitored at https://www.ataxia.org/pipeline/sca3/

[Post published on 03/05/2023 by Márcio Galvão]