Authors:

Takakazu Yokokura

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Spinal muscular atrophy (SMA) is a neuromuscular disease that is one of the leading causes of infant mortality. Mutations in the SMN1 gene cause the loss of motor neurons, leading to the muscle weakness that is characteristic of SMA. Fortunately, the Drosophila genome contains an ortholog of the SMN1 gene, Smn. In addition, defects in the neuromuscular junction (NMJ) are commonly seen in both SMA patients and the Drosophila SMA model, which make Drosophila an ideal model organism to study the genetics behind SMA. Receptor tyrosine kinases (RTKs) have been shown to affect the SMA phenotype. Most Drosophila studies have been conducted on RTK influence in neurons, not muscles, despite muscles being a significant part of the SMA phenotype. This study focuses on the phenotypic effects of inhibiting the Sevenless, Eph, Egfr, and Cad96Ca RTKs in Drosophila muscles. In past studies, these RTKs have been altered by the knockdown of Smn, prompting further study into their specific function at the NMJs. In the study, transgenic RNAi lines targeting each RTK, as well as a control, were selected and muscle-specific knockdown was induced. The third instar larvae were dissected, stained with anti-Dlg and anti-HRP antibodies, and imaged to obtain visuals of the NMJs on muscles six and seven in the A2 and A3 segments. The type 1B and 1S boutons, structures that regulate muscle contraction, in each image were counted. The bouton counts within each Drosophila line were aggregated and compared against the control. All four RTK genes exhibited statistically significant increased bouton counts, indicating that RTK inhibition affects NMJ structure and could alter the SMA disease phenotype. Future steps consist of investigating the specific function of RTK signaling in NMJ biology and the SMA phenotype. These results could have future clinical applications in treatments for patients with SMA. Optimizing the Sleeping Beauty Transposon System for Transgene Delivery in Hematopoietic Stem Cells Samuel Ha, Anna-Lena Neehus, Vijay Sankaran Harvard College | Currier House | Integrative Biology | 2027 B cells are central to human adaptive and humoral immunity and are generated by differentiation of CD34+ hematopoietic stem cells (HSCs) in the bone marrow. B cell differentiation can be modeled in vitro by directed differentiation of HSCs into B cell progenitors. However, lentiviral transgenes are often silenced during this process, limiting the possibility of pooled genetic screens to better understand biological mechanisms underlying B cell function and development. The Sleeping Beauty transposon system offers an alternative two-component transgene delivery approach in which the transposase enzyme excises a transposable element from a donor DNA template and integrates it randomly into the genome. However, both the transposon and transposase are typically delivered as plasmids, which has shown significant cytotoxicity in HSCs in vitro. To address this limitation, we utilized in vitro-transcription to develop a mRNA- based transposase. Delivery of the mRNA transposase along with a transposable element donor plasmid in HEK293T cells showed two-fold increased editing efficiency at the AAVS1 safe harbor locus in comparison to the plasmid-only system. In addition, transgene expression was increased and stable over three weeks of culture. To further reduce toxicity, we have engineered a transposable element plasmid to work with Cre recombinases, which can excise the transposable element as a minicircle plasmid. Minicircles are advantageous due to their small size and have been shown to lead to enhanced transgene delivery and lower cytotoxicity in a variety of cell types, including HSCs. This in parallel with the co-culture system will allow us to study the mechanisms that govern early B cell differentiation and how errors in the process can lead to malignancy. In total, our platform provides an alternative to lentiviral transgene delivery and provides a scalable toolkit to dissect B cell developmental mutations and accelerate therapeutic target discovery. Harvard Summer Undergraduate Research Village Program for Research in Science and Engineering

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Harvard / Economics and Molecular and Cellular Biology / 2028

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Co-authors:

Takakazu Yokokura