Safety and In Vivo Gene Delivery Efficiency of COATSOME® SS-Lipids, a Lipid Nanoparticle Platform Composed of Biodegradable Ionizable Lipids

Purpose: Messenger RNA (mRNA) therapeutics are a rapidly emerging treatment modality for a wide range of diseases. A common approach for mRNA therapeutics is to encapsulate in either a polymer or lipid nanoparticle to protect the nucleic acid against degradation and to cross the cell membrane for cytoplasmic delivery. Recently, ionizable lipid nanoparticles have emerged as viable carrier systems with one such lipid used in an approved product and multiple in clinical development. However these first generation ionizable lipids show significant dose limiting toxicity along with limited in vivo protein expression from mRNA therapeutics.

Here we report on the in vivo evaluation of a novel biodegradable ionizable lipid based LNP (COATSOME^® SS-OP) and its application to mRNA therapeutics and CRISPR-Cas9 gene editing.

Methods: LNP Preparation: The chemical synthesis of the SS-OP, SS-OB has been reported previously (1,2).  LNPs were prepared by the ethanol dilution method as described previously (4) with minor modifications. SS-OP LNP contained 52.5/7.5/40/3 mol ratio, whereas MC3 LNP contained MC3/DSPC/cholesterol/DMGPEG₂₀₀₀ at 50/10/38.5/1.5 mol ratio, respectively. In Vivo Studies: Particle size was measured by dynamic light scattering. Encapsulation efficiency of mRNA was assessed by the Ribogreen assay. The particle size of the LNP representing SS-OP, SS-OB, and MC3 were 86.5nm, 84.8nm, and 55.8nm respectively. Encapsulation efficiency ranged from 79% to 82 %. Repeat Dosing Study: C57BL6/J mice received tail vein injections of LNP equivalent to 0.05 mg/kg EPO mRNA. Injections were repeated every 7 days for up to 5 weeks. Blood samples were collected at 6h after each injection and analyzed by EPO ELISA.  Genome Editing Assay: C57BL6/J mice were administrated with the SS-OP LNP containing 0.375 mg/mg each of Cas9 mRNA and TTR sgRNA via the tail vein at Day1 and Day7. At Day 21 serum TTR was measured by ELISA and mice were sacrificed. Genome editing was investigated by PCR as described previously (3) of the extracted liver genome and percent editing was calculated by ratio of the digested and undigested bands on gel electrophoresis.

Results: Delivery of EPO mRNA to Hepatocytes: BALB/c mice were injected via tail vein of 0.05 mg/kg of mouse EPO-mRNA in LNP containing either SS-OP, SS-OB (a non-biodegradable analog of SS-OP)  or DLin-MC3-DMA (MC3). Serum EPO values, evaluated by ELISA, peaked at 3hrs to 1.2 x 10⁵ pg/ml (SS-OP), 5 x 10⁴ pg/ml (SS-OB) 4 x 10⁴ pg/ml (MC3), Single Dose Toxicity: Empty MC3 LNP were administered at 35 mg/kg and SS-OP administered at 35, 70, 175 and 350mg/kg total lipids to Sprague-Dawley SPF rats and hematology and blood chemistry were investigated on 1d post-administration. Liver enzyme elevation, was observed in the MC3 group and in the SS-OP group at 175 mg/kg and 350 mg/kg along with leukocytosis and hepatobiliary inflammation on pathology. No significant changes were observed in the SS-OP 35mg/kg, 70mg/kg, and control dose groups.

CRISPR-Cas9 Editing of TTR Gene: Two injections of the SS-OP LNPs containing both Cas9 mRNA and TTR sgRNA administered seven days apart resulted in a 95% decrease in the serum TTR concentration. Electrophoresis-based mutation assays indicated that 55% of the TTR-encoding genes in the mice liver were edited.

Conclusion: We demonstrated the potential for in vivo mRNA therapy with two proof-of-concept studies 1) therapeutic protein expression from liver and 2) gene editing. We also compared the activity and toxicity of SS-OP LNP against a reference compound, MC3. This lipid is the first and only ionizable lipid in an approved drug: patisiran. EPO mRNA delivered by SS-OP LNP provided for approximately 2.4X higher protein expression than delivery by the MC3 LNP.  Acute toxicity studies show SS-OP and MC3 lipids’ primary target organ is the liver, yet SS-OP can be dosed at significantly higher levels up to 175 mg/kg. These results suggest SS-OP lipids are a more potent, less toxic alternative to other ionizable lipids including MC3.

References:

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