Lipid nanoparticle delivery of TALEN mRNA targeting LPA causes gene disruption and plasma lipoprotein(a) reduction in transgenic mice

Figures

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  Figure 1. TALENs targeting human LPA gene (A) TALEN protein domain structure. (B) Genomic locus of human LPA and the binding locations of the designed TALENs, TAL1 and TAL2 (L, left; R, right). TAL1 targeted the KIV-2 domain of LPA and was custom designed using the TAL Effector-Nucleotide Targeter 2.0 web-based tool.20,21 Additional binding sites for TAL1 are indicated by the orange triangles and are due to the KIV-2 domain repeats present in the human reference genome. This illustration depicts the KIV-2 domain repeated seven times. TAL2 targeted the KIV-4 domain of LPA, and is an off-the-shelf prefabricated design from GeneCopoeia. (C) TALEN mRNA sequence design.

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  Figure 2. In vitro screening of LPA-targeting TALEN mRNAs (A) Representative immunofluorescence microscopy images of RT4 cells 16 h after transfection with 312 pM TALEN mRNAs. Scale bars, 75 μm. (B) Dose-dependent reduction of LPA transcripts measured by RT-qPCR 5 days after transfection of TALEN mRNAs in RT4 cells. The dotted line represents the LPA expression level in negative control mock transfected cells. (C and D) Capillary electrophoresis images of apo(a) protein level in RT4 whole-cell lysates 5 days following TAL1 (C) or TAL2 (D) mRNA transfection. (E) On-target gene editing analysis of RT4 cells 5 days following TALEN mRNA transfection. The frequency of frameshift-causing insertions and deletions (% Editing) was determined by next-generation sequencing of PCR amplicons from genomic DNA templates. For each condition, gDNA from n = 3 biological replicates was pooled and together used as the template for amplicon NGS gene editing analysis. (F) Schematic of TAL1 mRNA sequence optimization to generate TAL1 -opt. (G–I) In-cell western quantification of TAL1 protein level (G), representative capillary electrophoresis images of apo(a) protein level in whole-cell lysates (H), and…

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  Figure 3. LUNAR-TAL1 administration reduces Lp(a) and is well tolerated in a transgenic mouse model (A) Schematic representation of TAL1-opt mRNAs encapsulated in LUNAR LNPs (LUNAR-TAL1) administered intravenously (i.v.) in transgenic mice. (B) Schematic representation of the human LPA and human APOB transgenes used to create the double transgenic mouse model expressing human Lp(a). The human LPA transgene sequence was verified by Sanger sequencing (data not shown) and TAL1 target sites were confirmed, indicated with purple and red markers. (C) Schematic representation of study design. Mixed groups of male and female transgenic mice were given 0.5 or 2 mg/kg of LUNAR-TAL1 intravenously either once (1×) or twice (2×, 3 weeks apart). For all groups, plasma was collected at the indicated time points, and liver tissue was harvested at study termination (49 days after the first dose). (D) Lp(a) levels in plasma determined by a sandwich ELISA using a capture antibody recognizing apoB-100 and detection antibody recognizing apo(a). p > 0.05 (ns) and p < 0.0005 (∗∗∗) compared to PBS group, analyzed by two-way ANOVA with Tukey's multiple comparisons test. (E) Relative expression of LPA transgene in…

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  Figure 4. LUNAR-TAL1 administration in transgenic mice causes LPA gene disruption due to deletions at and between TAL1 target sites 1 and 2 (A) Schematic representation of human LPA transgene depicting editing sites for TAL1. Gene editing analysis (% Editing) at the corresponding target sites was determined by standard amplicon next-generation sequencing (NGS). p > 0.05 (ns) and p < 0.05 (∗) compared to PBS group, analyzed by one-way ANOVA with Tukey's multiple comparisons test. (B and C) Schematic representation of TAL1 target site 1 (B) and TAL1 target site 2 (C) at the DNA sequence level. Colored triangles indicate approximate locations of double-strand breaks caused by the FokI nuclease domain of TAL1. Red-shaded squares indicate bases at which there exists a mismatch between the reference sequence and the DNA-binding RVD in TAL1. (D) Representative subset of aligned reads generated from HiFi long-read sequencing of LPA transgene amplicons. Gray rectangles represent reads from PBS control mice, and purple rectangles represent reads from mice treated twice (2×) with LUNAR-TAL1 at 2 mg/kg. Black horizontal lines within reads represent sequence deletions. Reads are aligned to the LPA…