Manya
Gupta
98 Program for Research in Science and Engineering Understanding the Role of Receptor Tyrosine Kinases in Smn-Dependent Neuromuscular Junction Phenotypes in Spinal Muscular Atrophy
Abstract profile. Full document pending author claim.
Authors:
Manya Gupta, Takakazu Yokokura, David Van Vactor
Date Created:
2025-01-01
Course Title:
Professor:
Not specified
About Paper:
Spinal muscular atrophy (SMA) is a neuromuscular disease that of Smn, prompting further study into their specific function at the is one of the leading causes of infant mortality. Mutations in NMJs. In the study, transgenic RNAi lines targeting each RTK, the SMN1 gene cause the loss of motor neurons, leading to the as well as a control, were selected and muscle-specific knockdown muscle weakness that is characteristic of SMA. Fortunately, the was induced. The third instar larvae were dissected, stained with Drosophila genome contains an ortholog of the SMN1 gene, Smn. anti-Dlg and anti-HRP antibodies, and imaged to obtain visuals of In addition, defects in the neuromuscular junction (NMJ) are the NMJs on muscles six and seven in the A2 and A3 segments. commonly seen in both SMA patients and the Drosophila SMA The type 1B and 1S boutons, structures that regulate muscle model, which make Drosophila an ideal model organism to study contraction,ineachimagewerecounted. Theboutoncountswithin the genetics behind SMA. Receptor tyrosine kinases (RTKs) have each Drosophila line were aggregated and compared against the beenshowntoaffecttheSMAphenotype. MostDrosophilastudies control. All four RTK genes exhibited statistically significant have been conducted on RTK influence in neurons, not muscles, increased bouton counts, indicating that RTK inhibition affects despite muscles being a significant part of the SMA phenotype. NMJ structure and could alter the SMA disease phenotype. Future This study focuses on the phenotypic effects of inhibiting the stepsconsistofinvestigatingthespecificfunctionofRTKsignaling Sevenless, Eph, Egfr, and Cad96Ca RTKs in Drosophila muscles. in NMJ biology and the SMA phenotype. These results could have In past studies, these RTKs have been altered by the knockdown 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 a transposable element donor plasmid in HEK293T cells showed and are generated by differentiation of CD34+ hematopoietic two-fold increased editing efficiency at the AAVS1 safe harbor stem cells (HSCs) in the bone marrow. B cell differentiation locus in comparison to the plasmid-only system. In addition, can be modeled in vitro by directed differentiation of HSCs transgene expression was increased and stable over three weeks into B cell progenitors. However, lentiviral transgenes are of culture. To further reduce toxicity, we have engineered a often silenced during this process, limiting the possibility of transposable element plasmid to work with Cre recombinases, pooled genetic screens to better understand biological mechanisms which can excise the transposable element as a minicircle plasmid. underlying B cell function and development. The Sleeping Beauty Minicircles are advantageous due to their small size and have transposon system offers an alternative two-component transgene been shown to lead to enhanced transgene delivery and lower delivery approach in which the transposase enzyme excises a cytotoxicity in a variety of cell types, including HSCs. This transposable element from a donor DNA template and integrates in parallel with the co-culture system will allow us to study the it randomly into the genome. However, both the transposon mechanisms that govern early B cell differentiation and how errors and transposase are typically delivered as plasmids, which has in the process can lead to malignancy. In total, our platform shown significant cytotoxicity in HSCs in vitro. To address this providesanalternativetolentiviraltransgenedeliveryandprovides limitation, we utilized in vitro-transcription to develop a mRNA- a scalable toolkit to dissect B cell developmental mutations and based transposase. Delivery of the mRNA transposase along with accelerate therapeutic target discovery.
Abstract:
Spinal muscular atrophy (SMA) is a neuromuscular disease that of Smn, prompting further study into their specific function at the is one of the leading causes of infant mortality. Mutations in NMJs. In the study, transgenic RNAi lines targeting each RTK, the SMN1 gene cause the loss of motor neurons, leading to the as well as a control, were selected and muscle-specific knockdown muscle weakness that is characteristic of SMA. Fortunately, the was induced. The third instar larvae were dissected, stained with Drosophila genome contains an ortholog of the SMN1 gene, Smn. anti-Dlg and anti-HRP antibodies, and imaged to obtain visuals of In addition, defects in the neuromuscular junction (NMJ) are the NMJs on muscles six and seven in the A2 and A3 segments. commonly seen in both SMA patients and the Drosophila SMA The type 1B and 1S boutons, structures that regulate muscle model, which make Drosophila an ideal model organism to study contraction,ineachimagewerecounted. Theboutoncountswithin the genetics behind SMA. Receptor tyrosine kinases (RTKs) have each Drosophila line were aggregated and compared against the beenshowntoaffecttheSMAphenotype. MostDrosophilastudies control. All four RTK genes exhibited statistically significant have been conducted on RTK influence in neurons, not muscles, increased bouton counts, indicating that RTK inhibition affects despite muscles being a significant part of the SMA phenotype. NMJ structure and could alter the SMA disease phenotype. Future This study focuses on the phenotypic effects of inhibiting the stepsconsistofinvestigatingthespecificfunctionofRTKsignaling Sevenless, Eph, Egfr, and Cad96Ca RTKs in Drosophila muscles. in NMJ biology and the SMA phenotype. These results could have In past studies, these RTKs have been altered by the knockdown 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 a transposable element donor plasmid in HEK293T cells showed and are generated by differentiation of CD34+ hematopoietic two-fold increased editing efficiency at the AAVS1 safe harbor stem cells (HSCs) in the bone marrow. B cell differentiation locus in comparison to the plasmid-only system. In addition, can be modeled in vitro by directed differentiation of HSCs transgene expression was increased and stable over three weeks into B cell progenitors. However, lentiviral transgenes are of culture. To further reduce toxicity, we have engineered a often silenced during this process, limiting the possibility of transposable element plasmid to work with Cre recombinases, pooled genetic screens to better understand biological mechanisms which can excise the transposable element as a minicircle plasmid. underlying B cell function and development. The Sleeping Beauty Minicircles are advantageous due to their small size and have transposon system offers an alternative two-component transgene been shown to lead to enhanced transgene delivery and lower delivery approach in which the transposase enzyme excises a cytotoxicity in a variety of cell types, including HSCs. This transposable element from a donor DNA template and integrates in parallel with the co-culture system will allow us to study the it randomly into the genome. However, both the transposon mechanisms that govern early B cell differentiation and how errors and transposase are typically delivered as plasmids, which has in the process can lead to malignancy. In total, our platform shown significant cytotoxicity in HSCs in vitro. To address this providesanalternativetolentiviraltransgenedeliveryandprovides limitation, we utilized in vitro-transcription to develop a mRNA- a scalable toolkit to dissect B cell developmental mutations and based transposase. Delivery of the mRNA transposase along with accelerate therapeutic target discovery.
Source:
Harvard / Harvard College | Currier House | Mechanical Engineering | 2028 / 2025
Topics:
cell, sma, muscle, drosophila, transgene, plasmid, phenotype, rtk, system, delivery, transposable, element
