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14 Apr 2023, 9:43 am

Therapeutic strategies for autism: targeting three levels of the central dogma of molecular biology - Translational Psychiatry

The past decade has yielded much success in the identification of risk genes for Autism Spectrum Disorder (ASD), with many studies implicating loss-of-function (LoF) mutations within these genes. Despite this, no significant clinical advances have been made so far in the development of therapeutics for ASD. Given the role of LoF mutations in ASD etiology, many of the therapeutics in development are designed to rescue the haploinsufficient effect of genes at the transcriptional, translational, and protein levels. This review will discuss the various therapeutic techniques being developed from each level of the central dogma with examples including: CRISPR activation (CRISPRa) and gene replacement at the DNA level, antisense oligonucleotides (ASOs) at the mRNA level, and small-molecule drugs at the protein level, followed by a review of current delivery methods for these therapeutics. Since central nervous system (CNS) penetrance is of utmost importance for ASD therapeutics, it is especially necessary to evaluate delivery methods that have higher efficiency in crossing the blood-brain barrier (BBB).

This review has investigated three main targets of intervention for NDDs across the central dogma, specifically at the: DNA, mRNA, and protein levels. While there has been much success within in vitro and animal models at the DNA and protein levels, there has yet to be successful translation of these therapeutics in successful translation of these therapeutics in human clinical trials.

Efforts to treat gene haploinsufficiency will necessitate careful consideration of cell type specificity, tissue specificity, and modulating the expression of the correct splicing isoforms—something that will vary across tissues and developmental stages . Alternative splicing plays important role in NDDs, and ASD risk genes have differentially expressed isoforms (DEI) during every stage of prenatal development. Furthermore, DEIs impact pathways involved in dendrite development, synapse organization, and neuronal projection, the processes that are dysregulated in ASD. One study found that in NRXN1+/- human induced pluripotent stem cell (hiPSC)-derived neurons, there was a significant increase in expression of novel NRXNα isoforms, coupled with a decrease in the wild-type NRXNα isoform . This dysregulation in NRXNα isoform balance resulted in a reduction of neuronal activity and disruptions in neuronal maturation. Therefore, during the therapeutic development process, it is imperative to consider patient age, cell-type specific isoforms, alternative start sites, and alternative promoters for target genes. The strength of the promoter in transgene therapy or modulation of the CRISPRa transcriptional activators will play a key role in determining the appropriate treatment and avoiding unpredictable or undesired treatment consequences.

With the recent efforts of shifting focus to the therapeutic rescue of genes in NDDs, it may be possible that that the next decade will yield translational success. This success is dependent on further optimization of techniques still in their infancy such as CRISPRa or uORF-based/NMD-inhibiting ASOs. Furthermore, therapeutic mechanisms such as alternative splicing ASOs that have FDA approval for non-NDD pathologies such as Spinraza (SMA) and Eteplirsen (DMD) may provide a strong basis for further studies using these ASOs for genes with splice mutations implicated in NDDs. As evidenced by the mTOR inhibition therapeutics for TSC, the time point of therapeutic administration is also an important consideration for successful translation into clinic.

When it comes to NDDs, it will be of paramount importance to diagnose as early as possible in order to administer the therapeutics during the critical brain developmental periods. With this in mind, studies have shown that the late mid-fetal to early postnatal period is a critical window for neurodevelopment, in which many ASD risk genes are expressed .An example of such can be observed in a study where Cre-dependent activation of Ube3a in Ube3aStop/p+ embryonic mice restored all AS-associated motor, behavioral, and neurological deficits, whereas Ube3a reactivation in postnatal mice demonstrated diminishing efficacy with age for motor coordination rescue [183]. With the developing state of CNS-targeting therapeutic delivery strategies and the potential complications involved with delivering prenatal therapeutics, NDD’s are likely to be the most effectively treated during the early postnatal period. It is important to note that these time points will not be universally applicable for all genes and all phenotypes. Other studies have demonstrated successful rescue of neurological and electrophysiological deficits in adult Ube3aStop/p+ mice after reactivation of the Ube3a gene and rescue of neurological defects in adult Mecp2lox-Stop/y mice after reactivation of the Mecp2 gene. Similarly, the previously discussed Scn2a CRISPRa study successfully rescued electrophysiological deficits in adolescent Scn2a haploinsufficient mice [78]. This heterogeneity in phenotypic rescue across different developmental time points suggests that periods of therapeutic intervention will need to be assessed on a gene-to-gene basis.

With mouse models not being able to accurately recapitulate human neurodevelopmental periods, another translational step could be developing models of haploinsufficiency within NHPs—something that has already been established for the SHANK3 and MECP2 genes within cynomolgus macaques. The common marmoset (Callithrix jacchus) is another potential model organism for human NDD’s, with studies showing that the gene expression and gene distribution patterns within the brains of humans and marmosets were more similar than that of humans and mice . In addition to this, another study was able to successfully develop a transgenic marmoset model of Huntington’s disease (HD). These marmosets displayed dystonia and chorea—forms of involuntary movement that are physiological phenotypes of HD. Given these results, it may be valuable to pursue transgenic NHP models of NDD’s, with studies evaluating the efficacy of therapeutic treatments across different neurodevelopmental time points.

Another obstacle to clinical translation involves optimization of delivery methods for the therapeutics. When considering the use of viral vectors in NDDs, some important considerations include balancing tissue-specificity, immunogenicity, packaging limits, and ability to penetrate the BBB. With the advent of technologies such as lipid-based vectors, it may be possible to overcome the obstacles associated with the viral vectors for the development of therapeutics. However, since the lipid-based vectors are still in their infancy, further studies are needed to clearly compare both efficacy and safety between viral and lipid-based vectors.

A conversation with Lilia Iakoucheva and Derek Hong
There are no therapies approved for the core traits of autism, nor for most genetic syndromes linked to the condition. But there are many under investigation, and those that do come to fruition will likely target one of three levels of human biology, according to an expert review published in February in Translational Psychiatry: DNA, mRNA or proteins.

Such treatments could take the form of gene therapies, antisense oligonucleotides (ASOs) or small-molecule drugs, respectively, according to lead investigator Lilia Iakoucheva, professor of psychiatry at the University of California, San Diego, and Derek Hong, who worked on the paper as a graduate student in Iakoucheva’s lab. Spectrum caught up with Iakoucheva and Hong to discuss their ideas.

Spectrum: In the paper, you say it’s time to shift away from identifying more autism-linked mutations to doing something about treating them. Could you talk a little bit about that?

Lilia Iakoucheva:What I mean is that we should probably advance both fronts. Autism has many rare genetic variants associated with it, which means we have lots of low-hanging fruit, right? I did not mean to say we have to stop gene identification, but I think we have to also advance therapies. Whole-genome sequencing will help to identify the interplay between rare de novo and common inherited alleles. There is more and more evidence now that common inherited variants play a very important role in the genetics of autism, especially for the syndromes in which we cannot find a high-confidence rare de novo variant.

Derek Hong: To add on to that, I feel like recent whole-exome sequencing results drove a wedge into a door where we can begin to investigate therapeutics for those specific genes. But I think given more whole-exome sequencing investigation, we could potentially drive wedges through some more doors for targeting.

S: In this paper, you discuss three targets for intervention: DNA, mRNA and proteins. Which seems the most promising or furthest along?

The biggest things that come to my mind are the natural antisense transcripts that are expressed alongside certain autism susceptibility genes. Some natural antisense transcripts inhibit the translation of an autism-linked gene that’s implicated or that has a loss of function. I think it could be very promising to target these because ASOs themselves are relatively transient, meaning in a clinical setting we could potentially work with a dose that we know is tolerable and isn’t dangerous. And if necessary, after four months, when the drug is no longer in someone’s system, we can adjust that dosing without significant harm.

LI: Sometimes you can come up with treatment without even knowing the neurobiology. For example, in the ASO for spinal muscular atrophy (SMA), we still don’t know why those motor neurons are dying, but we have a treatment anyway.

But I think all three levels of intervention are valid, and they are all promising. CRISPR approaches should be approached with a high degree of caution because they’re harder to reverse, whereas small molecules are probably less dangerous because they will degrade. So in terms of development or safety, I think the small molecules will be the least dangerous, ASOs will be the second most and CRISPR-based approaches will be the most risky, in my opinion.

S: In terms of biological pathways, how do you decide which approach is most applicable?

It depends on what is being corrected, right? We know synapses and circuits are implicated in autism. But depending on the gene, there can also be translational pathways or ubiquitination pathways. You’re not going to correct everything with just one drug because autism is so multigenic. Even if a single gene is mutated, it still can influence a variety of different pathways. There is a lot of work that would need to be done to deconvolute all the pathways involved in autism.

S: In your paper, you talk about some of the obstacles yet to be overcome, including the blood-brain barrier and delivery challenges. What are the most significant obstacles, and what do you think it’s going to take to get past them?

The most challenging thing is to determine when to treat, what cell types to treat and how we judge whether the treatment actually helped. This may mean we need to select specific cohorts for clinical trials because maybe one treatment is not going to help everybody. There are lots of challenges to address before we can start treating, beyond things like the blood-brain barrier. Given so many different labs and companies working on delivery, I think that could be solved more easily than factors like at what time point and what population to treat, and how we judge improvement.

S: Are there models of success from the field?

Well, we have a very short list of Food and Drug Administration-approved therapeutics for genetic conditions so far. We have SMA, where the drug is delivered through a spinal tap. We have trofinetide, which was just approved for Rett syndrome.

DH: There are some phase 1 and 2 clinical trials for Angelman syndrome, specifically using ASOs to target the natural antisense transcripts for UBE3A. I’m watching that with some promise because there are three different groups working on that. That’s what I’m currently looking at. Especially given the ASOs used in the past for SMA.

[b[LI:[/b] Maybe the next one will be Angelman. Actually, it looks like that.

Professionally Identified and joined WP August 26, 2013
DSM 5: Autism Spectrum Disorder, DSM IV: Aspergers Moderate Severity

“My autism is not a superpower. It also isn’t some kind of god-forsaken, endless fountain of suffering inflicted on my family. It’s just part of who I am as a person”. - Sara Luterman


Joined: 5 Mar 2018
Gender: Male
Posts: 1,654

14 Apr 2023, 12:29 pm

Interesting stuff maybe the future and way to keep everyone happy is to stop calling it autism rather curing / treating individual impairments.

Even though autism is not a single condition & is made up of impairments to get a diagnosis its less politically charged

They should probably target ID first anyway

"The reasonable man adapts himself to the world; the unreasonable one persists in trying to adapt the world to himself. Therefore all progress depends upon the unreasonable man."

- George Bernie Shaw