Recovering canonical splicing in the SBDS gene to treat Shwachman-Diamond Syndrome

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Recovering canonical splicing in the SBDS gene to treat Shwachman-Diamond Syndrome

Shwachman-Diamond Syndrome (SDS) is a recessively inherited disease that affects patients' bone marrow health and white blood cell count, often leading to secondary illnesses like myelodysplasia and acute myeloid leukemia. Long-term damage to the central nervous system, skeletal muscles, and repetitive infections usually lead to premature death. The most common mutation in SDS patients disrupts a splice site (SS) in the SBDS gene. We hypothesized that the recently developed gene editing technique, Twin Prime Editing (TwinPE), could be used to insert a functional SS and correct SBDS splicing. Editing was initiated using plasmid delivery of the prime editor and two epegRNAs targeting an SBDStransgene in HEK 293Tcells. We had two designs for assessing editing efficiencies that consider either location of insertion site or insertion sequence content. We screened guides targeting different points within intron 2 that made different indellengths. Then, we tested insertion of sequences flanking the SS that were modeled after other genes' intronic sequences. The editing frequencies and splicing of SBDS were quantified using Sanger sequencing and qPCR. Using TwinPE, editing of the reporter led to over 200-fold increase in splicing, but expression was still lower compared to the original PE method inserting the same strong SS. Introduction of intronic sequences from other genes did not increase splicing further. These data suggest that TwinPE can be used to correct the SDS splice mutation. We plan to test TwinPE in primary HSPCs and will determine if these SBDS expression levels rescue the molecular defects of SDS.

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Record Origin: Recovering canonical splicing in the SBDS gene to treat Shwachman-Diamond Syndrome

Record Creation Date: 2023-05-16

Description Standard: MODS

Title: Recovering canonical splicing in the SBDS gene to treat Shwachman-Diamond Syndrome

Persons: Dipietro, Emma

Resource Type: Thesis/Dissertation

Date Created: 2023-04-27T00:00:00.000-04:00

Campus Location: N/A

Language: English

Extent and Media: PDF/OCR ; 3.71 MB

Physical Form: Color ; 37 pages

System of arrangement: DACS

Subject LCSH: Shwachman-Diamond Syndrome; Gene editing; Genetic disorders

Subject Local Topic: N/A

Biography or History: N/A

Custodial history: Transferred to the Emmanuel Archives from the author.

Acquisition Information: Transferred to the Emmanuel Archives from the author.

Appraisal Information: N/A

Accrual Information: Theses are collected on an annual basis in the spring semester

Conditions Governing Access: Access to document is governed by a license signed by the author

Conditions Governing Reproduction: Requests for permission to publish material from this thesis is governed by a license signed by the author. Contact the College Archivist.

Collection Title: Distinction in the Field: Senior Theses

Location of Originals: Folder: Dipietro_E

Alternative Form Available: N/A

Material Referenced: N/A

Referencing Material: N/A

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