AAV1-CMV-AsCpf1

Targeting key enzymes that produce oxalate precursors or substrates is another strategy to eliminate primary hyperoxaluria type I (PH1), the most common and most life-threatening type of primary hyperoxaluria. Compact clustered regularly interspaced short palindromic repeats (CRISPR) from Prevotella and Francisella 1 (Cpf1) proteins simplify multiplex gene editing and allow for all-in-one adeno-associated virus (AAV) delivery. The researchers hypothesized that the multiple functions of the Cpf1 system could help minimize oxalate formation in PH1 by simultaneously targeting the hepatic hydroxyacid oxidase 1 (Hao1) and lactate dehydrogenase A (Ldha) genes. The most potent and specific CRISPR RNA (crRNA) pairs targeting the rat Hao1 and Ldha genes were initially screened ex vivo. In vivo experiments demonstrated efficient genome editing of Hao1 and Ldha genes, leading mainly to small deletions. This results in reduced transcriptional and translational expression of Hao1 and Ldha. Treatment significantly reduced urinary oxalate levels, reduced kidney damage, and attenuated renal calcification in PH1 rats. No hepatotoxicity, ex-liver genome editing, or significant off-target effects were detected. Therefore, these studies demonstrate that the AAV-AsCpf1 system can target multiple genes and rescue the pathogenic phenotype in PH1, providing proof-of-concept for the development of gene therapies based on multiplex genome editing.

To assess the effects of treatment in vivo, researchers collected urine over a 24-hour period to quantify cumulative oxalate excretion. As shown in Figure 1A, urinary oxalate levels were consistently higher in Agxt^Q84X rats, approximately 3.0-fold higher than in age-matched WT rats. AAV-AsCpf1 treatment resulted in a significant reduction in urinary oxalate excretion in Agxt^Q84X rats over the 24-week experimental period. At 24 weeks post-treatment, both the WT and AAV-AsCpf1-treated Agxt^Q84X rats exhibited increased urinary oxalate excretion over the EG challenge period (Figure 1B). The kidneys in the PBS-treated group showed swelling and a significant increase in weight, approximately 1.7-fold that of AAV-AsCpf1-treated Agxt^Q84 rats and 2.0-fold that of WT rats (Figure 1D). Likewise, according to terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) fluorescence analysis (Figure 1E, F), compared with AAV-AsCpf1-treated Agxt^Q84 and WT control rats, the difference in PBS-injected Agxt^Q84 rats More apoptotic cells were detected. Furthermore, H&E staining of PBS-injected Agxt^Q84X rats revealed severe damage to renal tubular cells (Figure 1G). In contrast, Agxt^Q84X rats injected with AAV-AsCpf1 showed only mild tubular dilation and minimal calcium oxalate crystal deposition.

Figure 1. Therapeutic effects of Cpf1-mediated gene therapy. (Zheng R, et al., 2023)