(K-422) Lipid nanoparticle-mediated gene editing in mouse brain

Friday, October 13, 2023

3:30 PM – 4:30 PM PDT

Location: Exhibit Hall - Row K - Poster # 422

Presenting Author(s)

Sarah Cai, MS

PhD Student
Columbia University
New York, New York, United States

Co-Author(s)

CS

Cody Slater

MD PhD Student
Columbia University, United States

Presenting Author(s)

VY

Victoria Yang (any pronouns)

Undergraduate Student
Columbia University
Cambridge, Massachusetts, United States

Co-Author(s)

MP

Milan Patel

Undergraduate Student
Columbia University, United States
GG

Gabriel A. Gonzalez, n/a

Student
Columbia University
Ocala, Florida, United States
YZ

Yining Zhu

PhD student
Johns Hopkins University, United States
JM

Jingyao Ma, B.E. in Pharmaceutical Engineering from Wuhan University of Technology

M.S. Candidate
Johns Hopkins University, Department of Chemical and Biomolecular Engineering; Department of Materials Science and Engineering; Institute for NanoBioTechnology;, United States

Primary Investigator(s)

KL

Kam Leong

Professor
Columbia University, United States

Co-Author(s)

QW

Qi Wang

Professor
Columbia University, United States
HM

Hai-Quan Mao

Professor
Johns Hopkins University, Department of Biomedical Engineering; Department of Material Science and Engineering; Institute for NanoBioTechnology, United States

Introduction:: Genetic editing holds potential for a variety of applications such as treating neurological disease, understanding brain function, and repairing damage. However, one of the main challenges in brain gene editing is finding an efficient vehicle for delivering editing systems into the brain and the targeting of specific cells.
In this study, we used mRNA encoding Cre recombinase, as it strikes a balance between editing time and cost. The blood brain barrier (BBB), which serves as the main protective layer around the brain, must be bypassed. We use direct intracranial injection into brain tissue (Metzger et al., 2023). This involves the drilling of a small hole in the skull above the site of injection, then directly injecting into the brains of mice.
Genome editing in the brain via intracranial injection has been achieved using adeno-associated viruses (AAVs) (Staahl et al., 2017). An alternative, non-viral method of delivery is the use of lipid nano-particles (LNPs). For this study, LNPs were from the screen based on their ability to specifically transfect neuronal cells (higher neuronal cell transfection than in hepatocytes). LNPs were also selected for by size, in which only LNPs smaller than 200 nm are considered. Further, LNPs with similar compositions and transfection efficiencies are tested together in clusters of 3 LNPs.

Materials and Methods::

Preparation of Lipid Nanoparticles (LNPs)
A combination of helper lipids (DOPE, DOTAP, DSPC, 18PG), cholesterol, DMG-PEG2000 and Dlin-MC3-DMA were mixed at predetermined ratios. Cre mRNA was diluted in a 25 mM magnesium acetate buffer and added to lipid mixture while vortexing for a final concentration of 25 µg/mL. Trehalose was added to the filtered LNPs at a concentration of 9.5% w/v.LNP Characterization
The size and polydispersity index of LNPs were measured with DLS. mRNA concentration in LNPs were confirmed using nano spectrophotometry.
Intracranial Injection
Adult Ai14 mice were anesthetized by isoflurane gas and administered analgesics via subcutaneous injections. Stereotactic brain surgery was performed by first using a micromotor drill to create holes above the ventral posteromedial nucleus (VPM). Injections were done into the left and right VPM at 23 nL/s for a total of 1.86 µL. Following surgery, skin layers were closed with surgical glue and a postoperative cocktail of 5 mg/kg carprofen and 10 mg/kg Baytril were administered.Necropsy and Tissue Processing
One week post intracranial surgery, mice were anesthetized and transcardially perfused with PBS. Brain samples were trimmed using a rodent brain matrix centered around the injection site, then embedded in OCT compound and flash frozen. Brains were coronal sectioned into 10 µm slices and collected every 20-50 µm.Immunohistochemistry
Slices collected on glass slides were fixed with 4% PFA and washed three times in PBS. They were then coverslipped using Fluoromount-G with DAPI and stored in 4C until scanned.

Results, Conclusions, and Discussions:: Lipid nanoparticle (LNP) formulations synthesized were found to be within 80-200 nm in diameter with an average PDI of ~0.2. One week following intracranial injection of LNPs, Ai14 mice exhibited robust tdTomato signals, indicating successful genetic editing in the brain. The strongest signals were found along the injection path, with various levels of spread into the brain tissue. LNP cluster 2 was the most efficient at editing, achieving ~13% editing of the left brain hemisphere. This is significantly more than the signal quantified from the PBS control, which stands at ~1%.
The higher editing efficiency is likely due to its small size of approximately 90 nm, allowing for effective diffusion through the brain tissue matrix. Further quantification of LNP clusters could yield more results on the nanoparticle formulations that are the most efficient at achieving genetic editing delivery into the brain. This could have significant implications on future studies of the brain and in the development of potential treatment for neurodegenerative diseases. In conclusion, we demonstrated effective in vivo genetic editing using LNPs intracranially injected into the brains of mice.

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