Contributions
Abstract: EP1591
Type: Poster Sessions
Abstract Category: Therapy - Immunomodulation/Immunosuppression
As of 2018, the United States National Institutes of Health estimate that over half a billion people worldwide are affected by various autoimmune disorders. Though these conditions are prevalent, treatment options remain relatively poor, relying primarily on various forms of immunosuppression which carry potentially severe side effects and often lose effectiveness overtime. Given this, new forms of therapy are direly needed. We propose the use of small interfering RNA (siRNA) for the T-cell receptor β-chain gene (TRB) as a highly targeted, novel means of therapy for the treatment of autoimmune disorders. To meet this goal, the conducted research aimed to develop methods to produce siRNA for TRB. Additionally, this research included testing of said siRNA for its effects on the TRB gene and cell viability.
Jurkat (T-lymphocyte) cells were lysed and their mRNA isolated. After reverse transcription, C-tailing coupled with modified conventional techniques was applied in order to create siRNA specific for TRB. Additionally, siRNA synthesis using a previously reported multi-primer method was attempted (though was unsuccessful). Synthesized siRNA was subsequently coated with lipofectamine to facilitate transfection. The coated siRNA molecules were then applied to Jurkat cells at concentrations of 2 and 20 picomolar and incubated for 72h. Gene knockdown was quantified by qPCR, while cell viability was quantified both by direct counting and MTT assay.
While we were unable to synthesize siRNA for TRB using a previously reported “multi-primer method”, synthesis using C-tailing was successful. The encapsulated siRNA constructed in this manner demonstrated an ability to knockdown expression of TRB by more than 99.8% at concentrations of both 2 and 20 picomolar while having no observed effect on T-cell viability. This data suggests that siRNA for TRB may potentially be an effective, novel means of therapy in the treatment of autoimmune disorders.
Disclosure: Nicholas Magazine: nothing to disclose
Abstract: EP1591
Type: Poster Sessions
Abstract Category: Therapy - Immunomodulation/Immunosuppression
As of 2018, the United States National Institutes of Health estimate that over half a billion people worldwide are affected by various autoimmune disorders. Though these conditions are prevalent, treatment options remain relatively poor, relying primarily on various forms of immunosuppression which carry potentially severe side effects and often lose effectiveness overtime. Given this, new forms of therapy are direly needed. We propose the use of small interfering RNA (siRNA) for the T-cell receptor β-chain gene (TRB) as a highly targeted, novel means of therapy for the treatment of autoimmune disorders. To meet this goal, the conducted research aimed to develop methods to produce siRNA for TRB. Additionally, this research included testing of said siRNA for its effects on the TRB gene and cell viability.
Jurkat (T-lymphocyte) cells were lysed and their mRNA isolated. After reverse transcription, C-tailing coupled with modified conventional techniques was applied in order to create siRNA specific for TRB. Additionally, siRNA synthesis using a previously reported multi-primer method was attempted (though was unsuccessful). Synthesized siRNA was subsequently coated with lipofectamine to facilitate transfection. The coated siRNA molecules were then applied to Jurkat cells at concentrations of 2 and 20 picomolar and incubated for 72h. Gene knockdown was quantified by qPCR, while cell viability was quantified both by direct counting and MTT assay.
While we were unable to synthesize siRNA for TRB using a previously reported “multi-primer method”, synthesis using C-tailing was successful. The encapsulated siRNA constructed in this manner demonstrated an ability to knockdown expression of TRB by more than 99.8% at concentrations of both 2 and 20 picomolar while having no observed effect on T-cell viability. This data suggests that siRNA for TRB may potentially be an effective, novel means of therapy in the treatment of autoimmune disorders.
Disclosure: Nicholas Magazine: nothing to disclose