Introduction
The pathology of many genetic central nervous system (CNS) diseases begins before birth, resulting in significant morbidity by the time of birth or shortly after, as reviewed elsewhere. Prenatal DNA sequencing allows the identification of disease-causing mutations to treat disease in utero before birth. Use of mRNA-based gene editing tools, including CRISPR-Cas9 and base editing platforms, provides an opportunity for “one-and-done” treatments for monogenic diseases (Doudna 2020).
In addition to preventing the onset of disease pathology, in utero gene editing of small fetuses maximizes the therapeutic dose per fetus weight and leverages accessible and abundant fetal stem/progenitor cells, thus supporting the persistence of the therapeutic edit. Also, the tolerant fetal immune system minimizes an immune barrier to gene editing tools.
As outlined in this blog, Palanki et al. (2023) developed lipid nanoparticles (LNPs) for delivery of mRNA base-editing platforms to fetal mouse and monkey brain. This remarkable proof-of-concept used TriLink CleanCap® 5-methoxyuridine (5moU)-modified Cre recombinase to establish brain-cell selectivity prior to delivery of base-editing mRNA, co-transcriptionally synthesized with CleanCap® AG analog and N1-methylpseudouridine-5’-triphosphate from TriLink.
This publication by Palanki et al., which was co-authored by 2023 Nobel Laureate Drew Weissman, details the interdisciplinary research of a 28-member team. Selected findings highlighted herein are organized in the following sections:
- Synthesis and Screening of LNPs for Brain Delivery
- Cre mRNA-LNP Gene Modulation in Periventricular Cells of Mouse Brain
- Optimizing LNPs for Co-Delivery of Base-Editing Technology
- Mutation Correction by Adenine Base Editor mRNA Delivery in Mouse and Monkey
- Clinical Potential in Humans
(1) Synthesis and Screening of LNPs for Brain Delivery
It is known that branched-tail ionizable lipid-containing LNPs potently deliver mRNA in vivo due to enhanced ionization at late endosomal pH 5 that facilitates intracellular release of mRNA into the cytoplasm for translation into protein (Hajj et al. 2019). Palanki et al. therefore synthesized a structurally diverse library of 12 branch-tail ionizable lipids by reacting 3 different length terminal-epoxide alkanes with 4 polyamines. Each of these 12 candidate lipids was separately mixed with the widely used phospholipid abbreviated as DOPE, cholesterol, and a lipid-anchored polyethylene glycol (PEG) at ratios determined by previous optimization of tumor vaccines in adult mice.
The resultant formulations were each mixed with firefly luciferase (Fluc) mRNA to obtain a library of 12 Fluc-reporter LNPs for screening. Delivery to the brain in perinatal (i.e., fetal and neonatal) mice involved administration by intracerebroventricular (ICV) injection of fetuses in a neuro-developmental stage akin to a mid-gestation human fetus, a time point when clinical therapeutic intervention is technically feasible. The ICV injection route was selected because it has been reported to be clinically safe and effective for delivery of therapeutic agents for a broad range of neurological diseases.
Fetuses were assessed 4 h after ICV injection for Fluc expression using bioluminescence imaging (BLI) of whole fetus and individually dissected fetal brains. The top-performing LNP, termed C3 LNP, resulted in 17-fold greater mRNA expression in the fetal brain compared to an FDA-approved, industry-standard control.
Notably, BLI-based biodistribution of C3 LNPs showed strong Fluc expression in the fetal brain but no significant expression in other organs, indicating brain specificity. Shortly after birth, BLI of neonatal brains confirmed the performance trends observed in the fetal brain, with C3 LNPs facilitating 8-fold greater mRNA expression in the neonatal brain than the industry standard LNP. These results supported the use of neonatal mouse models in subsequent studies, greatly facilitating experimentation compared to fetal mice in utero.
(2) Cre mRNA-LNP Gene Modulation in Periventricular Cells of Mouse Brain
To gain insight into cellular selectivity of LNP-mediated mRNA delivery in neonatal brain, Cre-reporter mice (termed mT/mG mice) were used. These mice constitutively express red-fluorescent, membrane-targeted Tomato (mT) protein, absent Cre recombinase-mediated excision allowing expression of membrane-targeted green fluorescent protein (mG).
Brain biodistribution was measured for high- (C3), mid- (C2), and low-performing (C1) ionizable LNPs containing TriLink Cre recombinase 5moU-modified mRNA, after ICV administration to postnatal day-0 mT/mG mice. One week after injection, harvested neonatal brains were analyzed for gene modulation via tissue histology. The found magnitude of mG expression was proportional to LNP performance in the initial library screening studies (C3 > C2 > C1).
C3 LNP-treated neonates did not show significant diffusion deep into the brain, but instead exhibited strong mG expression in the periventricular areas and adjacent cortical structures. Flow cytometry and cell-specific brain histology confirmed mG expression in microglia lining the ventricles, astrocytes, and neurons with limited penetration to internal brain structures.
Importantly, efficient targeting of periventricular cells offers potential to produce therapeutic proteins for secretion into cerebrospinal fluid (CSF) circulation and utilize paracrine (i.e., nearby) cell-signaling effects for treatment of disease.
(3) Optimizing LNPs for Co-Delivery of Base-Editing Technology
While the C3 ionizable lipid is a major determinant of intracellular delivery by both directly complexing with mRNA and facilitating endosomal escape, the other excipients also play critical roles: DOPE supplies structural support, cholesterol provides stability and enables membrane fusion, and PEG reduces aggregation and nonspecific endocytosis.
To optimize these parameters for C3 LNP-mediated delivery for base editing brain cells, 27 LNP formulations were screened for delivery of green fluorescent protein (GFP) mRNA to mouse neuroblasts. An optimal ratio of components was thus identified and then tested with a range of ratios of ionizable lipid (N) and nucleic acid phosphate (P). This led to selection of a 10:1 N:P ratio for all further studies.
Next studied was the transition from GFP mRNA-optimized C3 LNPs to co-delivery of CRISPR base-editing mRNA and single-guide RNA (sgRNA) to perinatal brain cells. For this, Palanki et al. co-encapsulated both adenine base editor (ABE7.10) mRNA (made with CleanCap® AG analog and N1-methylpseudouridine-5’-triphosphate from TriLink) and an sgRNA specific for the Idua G → A (W392X) mutation present in the mouse model of mucopolysaccharidosis type I (MPS-IH, Hurler syndrome). This co-encapsulated MPS treatment was termed C3.MPS LNPs.
C3.MPS LNPs encapsulating ABE7.10 mRNA and sgRNA at 3 different mass ratios were formulated and assessed for on-target editing in MPS-IH mouse fibroblasts. A 3:1 ratio of ABE7.10 mRNA : sgRNA gave the highest level of editing and was used in a dose-response study for on-target editing in MPS-IH mouse neurons, as well as toxicity assessment in MPS-IH mouse fibroblasts and neurons. These studies demonstrated dose-dependent A → G editing of the target adenine with no toxicity.
(4) Mutation Correction by Adenine Base Editor mRNA Delivery in Mouse and Monkey
Mouse Model: Palanki et al. next investigated whether durable biochemical corrections resulted in the brains of Idua-W392X mice treated with C3.MPS LNPs. Like patients with MPS-IH, Idua-W392X mice have undetectable α-L-iduronidase (IDUA) enzyme activity and elevated tissue glycosaminoglycans (GAGs). At one month, mice treated with C3.MPS LNPs demonstrated increased IDUA activity in the midbrain (7% of normal) and hippocampus (11% of normal), relative to untreated Idua-W392X mice. This was associated with a reduction in GAG levels in the respective brain regions.
These data suggest that C3.MPS LNPs facilitate partial biochemical correction of disease in the neonatal Idua-W392X mouse brain, demonstrating the potential for mitigation of brain disease in MPS-IH.
The genomic safety of this LNP-mediated base editing strategy was investigated by sequencing of brain genomic DNA. Edited mice did not demonstrate off-target base editing above background at 9 previously identified possible off-target sites for the sgRNA targeting the mouse Idua G → A mutation.
Monkey Model: The clinical potential of this delivery platform was evaluated in macaques at a gestational age that approximates the developmental stage of a mid-gestation human fetus when therapeutic intervention is clinically feasible.
C3 LNPs encapsulating GFP mRNA were prenatally administered ICV in utero and, 48 h after injection, fetuses were delivered by caesarian section and brain GFP expression was assessed by immunohistochemistry. Tissue sections of harvested fetal macaque brains demonstrated strong GFP expression in the cells lining the lateral ventricles, analogous to the region transfected in the mT/mG mouse studies.
This result demonstrates that the mRNA delivery performance of C3 LNPs in the perinatal brain translates to a larger scale animal model more developmentally akin to a human, while highlighting the need for future optimization to enhance targeting deeper regions of the NHP brain.
(5) Clinical Potential in Humans
To demonstrate C3 LNP-mediated base editing in human brain tissue, slices of pediatric brain tissue were cultured ex vivo in a 50:50 mixture of human CSF and serum-free media. C3 LNPs encapsulating cytosine base editor (BE3) mRNA and an sgRNA to introduce a C → T mutation (W402X) in the IDUA gene (a common mutation in MPS-IH patients) were applied to the slices. Sequencing demonstrated ∼4% on-target editing, supporting the ability of C3 LNPs to co-deliver a functional base editor mRNA and sgRNA in an ex vivo human brain tissue model.
Concluding Comments
This proof-of-concept study supports the safety and efficacy of optimized LNPs for the delivery of mRNA-based gene editing therapies to the CNS. The lead LNP developed in this study holds promise for CNS diseases in which a paracrine effect of restoring normal protein function is adequate, following periventricular cell targeting. Palanki et al. concluded that further LNP optimization is required to apply this delivery modality to a broader range of diseases involving non-periventricular structures or specific neural cell types.
Your comments are welcomed, as usual.
Please feel free to share this blog with your colleagues or on social media.
