Poster J4 - Poly(beta-amino ester)-based Nanoparticles Allow For In Vivo Gene Delivery To Cardiomyocytes

Friday, October 25, 2024

3:30 PM – 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Lance Xu

Undergraduate Research Assistant
Johns Hopkins University, United States

Co-Author(s)

EY

Edwin Yoo

Cardiology Fellow
Department of Cardiology, Johns Hopkins Hospital, United States
AS

Alberto Sendagorta

Scientist
Vertex Pharmaceuticals, United States
MW

Marcus Williams

MD/PhD Candidate
University of California, San Fransisco, United States
NK

Navid Koleini

Postdoctoral Fellow
Department of Cardiology, Johns Hopkins Hospital, United States
Stephany Y. Tzeng, PhD

Assistant Research Professor
Johns Hopkins University, United States

Primary Investigator(s)

Jordan J. Green, PhD (he/him/his)

Professor
Johns Hopkins University, Maryland, United States

Introduction: Heart failure (HF) is a leading cause of death worldwide for which there is no cure, and approximately 1 in 4 people in the US will develop it in their lifetime. The majority of heart disease ultimately results in cardiomyocyte (CM) dysfunction which is largely responsible for heart failure. While the causes of HF are varied across a broad spectrum, the transcriptional signatures across CMs are consistent, allowing precision targeting of select genes to improve HF outcomes. Some genes, such as sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a), have already shown potential in pre-clinical studies to treat HF, however, gene delivery systems such as via adenovirus have failed in late-stage clinical trials, likely due to low transfection efficiency.

PBAE (poly(beta-amino ester)) polymers are cationic and biodegradable polymers that have been engineered to deliver to and allow cells to internalize genetic material without significant loss in transfection efficiency and are not immunogenic, unlike viral-based therapies. PBAEs require little effort to synthesize, allowing high throughput time-efficient development of PBAE libraries that can be easily screened to fine tune efficiency and targeting of specific tissues. In this study, we engineered a specific PBAE termed “4-5-6” that was found to have tropism towards the heart and successfully transfected CMs with plasmid DNA. These findings will allow for downstream gene therapy applications with DNA plasmids encoding the Cas9 enzyme and specific guide RNA.

Materials and

Methods: PBAEs are composed of three components: backbone, sidechain, and endcap. They were synthesized via Michael addition reactions (Figure 1a), which allows for the efficient synthesis of thousands of combinations of polymers. Several polymers were selected for screening of viability and GFP in vitro via flow cytometry (Figure 1b). Nanoparticles (NPs) were formed by adding polymers to DNA plasmid encoding GFP protein and transfected to extracted neonatal ventricular rat cardiomyocytes (NVRMs). The highest performing polymers from this initial screening were selected for further in vitro (Figure 1c, 1g) and in vivo studies (Figure 1d-f). Mice 8 weeks old were injected retro-orbitally with 25μg of DNA plasmid encoding luciferase in a 40 weight/weight (Polymer/DNA) ratio with 9mg sucrose as the excipient. D-luciferin was injected into the mice subcutaneously at 6 hours and imaged for luminescence using an IVIS imaging system (Figure 1d).

To verify CM transfection, we utilized an Ai9 mice model (Figure 1e). Successful transfection of DNA plasmid encoding Cre recombinase allows the enzyme to cut between loxP sites in the genome containing a stop codon, resulting in the permanent expression of fluorescent protein tdTomato. CMs were then extracted from the mice and imaged using fluorescent confocal microscopy (Figure 1f).

Results, Conclusions, and Discussions: The screening of PBAE polymers in vitro showed the 456 polymer as the best balance between high transfection efficiency and viability (Figure 1a-b). Further in vitro analysis of transfection in NVRM cells revealed an increasing dose-response to 456 NPs (Figure 1c), where 536 was used as a positive control. B16-F10 cells were also analyzed for dose-response of 456, but transfection efficiency dropped at 60 and 90w/w due to toxicity (Figure 1c).

456 was found to have a tropism towards the heart and lung after transfection of mice with luciferase DNA (Figure 1d). Further, CMs specifically were found to be transfected (Figure 1f) in an Ai9 mice model transfected with Cre recombinase. Preliminary studies conducted to compare transfection efficiency in CMs revealed a higher transfection efficiency compared to the industry standard, Lipofectamine 3000 (Figure 1g).

These results show that 456 has a tropism towards the heart specifically and can transfect CMs within the heart. 536, a historically well performing polymer and shown to have comparable transfection in vitro, has a tropism towards the liver and not the heart (data not shown). The discovery of the unique tropism of 456 to the heart is particularly exciting for heart failure therapies. Current approaches of gene delivery to the heart often fail due to low transfection efficiency ( < 2% transfection), but the development of a high efficiency PBAE with tropism towards the heart will provide a solution to the current setbacks in heart gene delivery.

Further experiments will clarify the percentage of CMs transfected in vivo via flow cytometry, which is challenged by the elongated shape of CMs. Additionally, sequential delivery of Cre recombinase DNA will be tested to assess whether a cumulative repeat dosing strategy can be effective for in vivo delivery.

Acknowledgements (Optional): I would like to thank Dr. Edwin Yoo for his continued support and mentorship throughout this project, Dr. Green for allowing us to use his lab space and equipment to facilitate this project, and all the Green Lab members who helped me troubleshoot specific protocols in this project.