While ultrasound-mediated gene delivery (UMGD) has been accomplished using high peak negative pressures (PNPs) of 2 MPa or above， emerging research showed that this may not be a requirement for microbubble (MB) cavitation. Thus， we investigated lower-pressure conditions close to the MB inertial cavitation threshold and focused towards further increasing gene transfer efficiency and reducing associated cell damage. We created a matrix of 21 conditions (n = 3/cond.) to test in HEK293T cells using pulse durations spanning 18 μs-36 ms and PNPs spanning 0.5-2.5 MPa. Longer pulse duration conditions yielded significant increase in transgene expression relative to sham with local maxima between 20 J and 100 J energy curves. A similar set of 17 conditions (n = 4/cond.) was tested in mice using pulse durations spanning 18 μs-22 ms and PNPs spanning 0.5-2.5 MPa. We observed local maxima located between 1 J and 10 J energy curves in treated mice. Of these， several low pressure conditions showed a decrease in ALT and AST levels while maintaining better or comparable expression to our positive control， indicating a clear benefit to allow for effective transfection with minimized tissue damage versus the high-intensity control. Our data indicates that it is possible to eliminate the requirement of high PNPs by prolonging pulse durations for effective UMGD in vitro and in vivo， circumventing the peak power density limitations imposed by piezo-materials used in US transducers. Overall， these results demonstrate the advancement of UMGD technology for achieving efficient gene transfer and potential scalability to larger animal models and human application.