Lead Members: Renee Napoliello, Katie Lucot, Paula Vij, Julian Halmai

CRISPR ACTIVATION THERAPY FOR ADNP, PITT-HOPKINS, and FOXG1 SYNDROME

Description: In some cases, when one of your two gene copies is mutant, not enough functional gene product is made for the cell – this is haploinsufficiency. We are researching CRISPR activation (CRISPRa) therapy to target the promoter of the wild type gene to increase expression of the wild type allele. One way of delivering our therapy to neurons is using a viral capsid to inject DNA coding the therapy. Due to capsid size limitation, The Fink lab has developed a “split dCas9” plasmid system (sp-dCas9) to code our therapy into separate capsids.

CRISPR ACTIVATION THERAPY FOR SYNGAP1 SYNDROME

Description: Paula works in the characterization and cell modeling of a genetic disorder on a gene called SYNGAP1 using patient-specific iPSC-derived cells. This gene provides instructions for making a protein called SynGAP1, which plays an important role in the brain. SynGAP1 helps regulate synapse adaptations and promotes proper brain wiring, which is particularly important during a critical period of early brain development that affects future cognitive ability. De novo mutations in this gene result in loss of functions and cause haploinsufficiency, leading to MRD5, whose phenotype comprises intellectual disability, developmental delay, autism, and epilepsy. The goal of her project is to use a genome modifying mechanism, CRISPR-mediated activation (CRISPRa), which targets a transcriptional activator to the gene’s regulatory element as a viable approach that could potentially correct SYNGAP1 haploinsufficiency.

GENE THERAPY AS POTENTIAL TREATMENT FOR ADNP SYNDROME

Description: A second potential therapeutic for ADNP syndrome is delivering additional copies of the wild type allele to the nucleus for expression. Thus additional transcripts of the “healthy” gene are available for protein translation. We are researching injecting cDNA of the ADNP gene into cells for that end.

ANTISENSE OLIGONUCLEOTIDES AS POTENTIAL TREATMENT FOR ADNP SYNDROME

Description: A third potential therapeutic for ADNP syndrome involves antisense oligonucleotides. These small pieces of RNA bind to false translational start sites on the mRNA of the transcript, thereby increasing translation efficiency and expression of the wild type allele.

EPIGENETIC EDITING AS POTENTIAL THERAPY

Lead Member: Julian Halmai

Summary: CDKL5 deficiency is a rare intractable epilepsy that is caused by mutations in the CDKL5 gene on the X chromosome. In females one of the two X chromosomes becomes randomly inactivated. This results in a mosaic of tissue with cells expressing either the wildtype or mutant allele. In cells expressing the mutant allele, this leaves an epigenetically silenced wildtype allele behind that when reactivated could potentially rescue phenotypes associated with the disorder. We are utilizing patient-derived stem cell and transgenic mouse models to test our therapeutic interventions. Our lab is also heavily focused on Rett syndrome, in which we are applying similar strategies for reactivation of the MECP2 gene in female somatic cells as part of a bigger collaboration with the Liu lab at Columbia University and the Bedalov lab at Fred Hutch (press release here). The goal is to expand this strategy to many other X-linked intellectual disabilities with an underlying loss of function mutation, such as CASK-related intellectual disability.

RNA EDITING AS POTENTIAL THERAPY

Lead Member: Jasmine Carter

Summary: Jordan’s Syndrome is a rare genetically-linked intellectual disability resulting from de novo heterozygous missense mutation in PPP2R5D. Our lab utilizes Jordan’s Syndrome patient-derived fibroblasts to develop induced pluripotent stem cell (iPSC) derived neuronal disease models to understand the underlying molecular and cellular mechanisms associated with variants in PPP2R5D. We are employing the novel CRISPR/dCas13 RNA-targeting system to edit pathogenic adenosine nucleotides in the PPP2R5D transcript in these in vitro cellular models

PRIME EDITING AS POTENTIAL THERAPY

Lead Member: Isaac Villegas

Summary: Prime editing is a variation of the traditional CRISPR-Cas9 editing. A nickase cuts one of the two strands of DNA at a target site. Then a reverse transcriptase reads off an RNA template (attached to the guide RNA) to replace one of the strands of DNA at the site. The cell naturally resolves the mismatch, sometimes incorporating the edit. This could be harnessed to target some mutations in PPP2R5D that cause Jordan’s syndrome. By targeting a prime editer to the site of the mutated sequence, we can potentially insert the correction.

CRISPR INTERFERENCE AS POTENTIAL THERAPY

Lead Member: JJ Waldo

Description: Huntington’s Disease involves the pathogenic CAG expansion of the HTT gene. Huntington’s disease is typically inherited, and there are cases where the promoter is also inherited as a haplotype block. Unique SNPs in the promoter of the mutant allele can thus be targeted by a guide RNA for CRIPSR interference, thereby downregulating expression.

PROTEIN BIOMARKERS FOR ANGELMAN’S SYNDROME

Lead Member: David Cameron

Summary: I am investigating protein biomarker targets for Angelman Syndrome that may be used to determine efficacy of treatment and rescue of phenotype. I am also evaluating the effect of a small molecule therapy that shows potential for reducing neuroinflammatory response associated with neurodegenerative disorders.

Lead Members: Klaudia Braczyk, Julian Halmai

We are applying similar strategies for Rett syndrome as CDKL5 deficiency disorder for reactivation of the MECP2 gene in female somatic cells. This involves the reactivation of the wild type allele on the epigenetically silenced X chromosome. This project acts as part of a bigger collaboration with the Liu lab at Columbia University and the Bedalov lab at Fred Hutch (press release here). The goal is to expand this strategy to many other X-linked intellectual disabilities with an underlying loss of function mutation, such as CASK-related intellectual disability.

Lead Members: Casiana Gonzalez

Description: My project focuses on optimizing our current epigenetic CRISPR/dCas9 platform to meet AAV packaging criteria and applying this system towards CASK-Related Intellectual Disability. Further, I plan to assess this system in patient relevant cells, such as iPSC-derived neurons, and measure functional rescue from the disease phenotype. This project allows for a therapeutic platform that can be packaged in a clinically relevant AAV delivery method, as well as insight on function post-treatment.