Cutting through the stones: Unlocking therapeutic potential with gene editing tools for primary hyperoxaluria type 1

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Primary hyperoxaluria type 1 (PH1) is a severe metabolic disorder caused by a deficiency in the alanine-glyoxylate aminotransferase (AGT) enzyme responsible for catalyzing the glyoxylate-to-glycine conversion. When AGT is impaired, glyoxylate is converted to oxalate instead, which cannot be metabolized effectively and forms insoluble calcium oxalate crystals. These crystals accumulate as kidney stones in the urinary tract and kidney tissue, progressively impairing kidney function and affecting other organs over time. Without treatment, PH1 can be life-threatening.¹ Traditional treatment approaches have focused on supportive therapies, including hyperhydration and crystallization inhibitors. Vitamin B6 (pyridoxine), a cofactor for AGT, can significantly reduce oxalate production in about 20%–30% of PH1 patients who are B6 responsive. In the most severe cases, isolated kidney or combined liver/kidney transplantation may be considered; however, transplants carry significant risks of morbidity and mortality.² Recent RNA interference (RNAi)-based therapies have been developed to reduce enzymatic substrates and lower oxalate production. In PH1, two hepatic enzymes have been targeted: glycolate oxidase (GO) with lumasiran and lactate dehydrogenase (LDH-A) with nedosiran. However, these treatments require repeated dosing and are costly. This highlights the unmet need for durable, minimally invasive, and cost-effective therapies for PH1. In vivo gene editing with CRISPR-Cas9 offers a promising alternative by disrupting GO enzyme expression to reduce oxalate production.

In a new study published in this issue of Molecular Therapy, Jiang et al.³ sought to develop a gene-editing strategy to treat PH1 by delivering CRISPR-Cas9 in vivo using lipid nanoparticles (LNPs) for GO disruption. To test the potency of the treatment, they additionally created a PH1 mouse model by knockdown of Agxt. Previously, an effort to create a mouse model of PH1 by CRISPR-Cas9 focused on gene editing of murine zygotes.⁴ Agxt^−/− mice inconsistently displayed a disease phenotype, even when stressed by the addition of ethylene glycol (EG) in drinking water. Here, the authors were able to develop a model of PH1 that consistently demonstrated the disease phenotype. After intravenous injection of LNP containing Cas9 mRNA and Agxt-targeting single-guide RNA (sgRNA) in wild-type (WT) C57BL/6 mice, injected mice subjected to metabolic stress induced by the addition of 0.5% EG in drinking water exhibited elevated urinary oxalate levels and kidney CaOx deposits. The authors were then able to show correction of this phenotype by CRISPR-Cas9-mediated inhibition of GO using sgRNA targeting the murine hydroxyacid oxidase 1 (mHao1). Two weeks after injecting mice with LNP-CRISPR-Cas9-Agxt, PH1 mice were injected with Cas9 mRNA and sgRNA targeting mHao1 encapsulated in LNP (LNP-CRISPR-Cas9-mHao1). The mice exhibited durable editing at both Agxt and Hao1 up to 12 months post-injection and displayed significant reductions in urinary oxalate levels when treated with both LNPs 5 months after treatment. Treatment with the LNP-CRISPR-Cas9-mHao1 also protected mice from developing CaOx deposits after being challenged with EG for four weeks. The beneficial effects seen from mHao1 editing were able to be sustained after liver regeneration. The authors then generated a humanized hHAO1 knockin mouse model by knocking in a modified hHAO1 sequence into murine zygotes to explore whether treatment of PH1 through hHAO1 gene editing could be plausible. hHAO1-KI mice were treated with LNP-CRISPR-Cas9-Agxt and exposed to 0.5% EG in drinking water to induce the disease phenotype. They were then administered an LNP containing Cas9 and sgRNA targeting the human HAO1 gene. Treated mice showed reduced oxalate levels in urine, indicating that this strategy can alleviate the effects of the disease. The authors therefore demonstrate a powerful new approach for the treatment of PH1 that provides an alternative to the multi-dose genetic therapies currently available.

In vivo delivery of gene-editing components enables precise genetic modifications, offering a gold-standard therapy for inherited diseases by permanently correcting pathogenic mutations with a single application. Early applications of gene editing are best suited for diseases treatable through gene disruption rather than gene correction, as DNA repair mechanisms without nucleotide insertion are more efficient. PH1 is a strong candidate for genome editing and oligonucleotide therapies, as gene knockdown or knockout of the hepatic enzymes GO and LDH-A provides significant therapeutic benefits. Small interfering RNAs (siRNA) effectively silence gene expression through RNAi, and this powerful modality has already been harnessed into a US Food and Drug Administration (FDA)-approved drug for PH1, lumasiran.⁵ Lumasiran degrades the mRNA that encodes GO, leading to reduced urinary oxalate excretion. The strategy presented in the Jiang et al. study similarly involves the elimination of GO expression, but with the additional benefit that only a single dose should be required to achieve durable therapeutic benefit. Several preclinical studies have investigated CRISPR-Cas9-based gene-editing strategies for treating PH1, with most utilizing adeno-associated virus (AAV) vectors for delivery. However, these studies still face certain limitations (Table 1).^3,6,7,8,9 While no FDA-approved drugs currently use LNP delivery of CRISPR-Cas9, clinical programs are exploring this approach for treating transthyretin amyloidosis,¹⁰ a condition already addressed by an FDA-approved siRNA therapy.¹¹ Ongoing trial results will help determine whether CRISPR-based mRNA therapies or siRNA therapies offer greater therapeutic potential.

Table 1.

The preclinical gene-editing strategies for treating primary hyperoxaluria type 1

Publication title	Authors	Gene-editing approach	Therapeutic Goal	Model/system used	Key findings	Challenges/limitations
CRISPR-Cas9-mediated glycolate oxidase disruption is an efficacious and safe treatment for primary hyperoxaluria type 1	Zabaleta et al., 20186	Systemic administration of a liver-specific AAV8-SaCas9-Hao1-sgRNA (sgRNAs g1 + g2)	Disruption of mHao1	PH1 mouse model (Agxt1−/− mice)	48% (g1)–52% (g2) liver indels; prevention of renal oxalate accumulation, reduced oxalate excretion in urine, reduced oxalate crystal formation in kidneys	Off-target, continuous Cas protein expression from the AAV vector, insertion of AAV at CRISPR cut site, therapeutic benefit with multiple enzyme knockouts, optimization of editing efficiency
In vivo CRISPR-Cas9 inhibition of hepatic LDH as treatment of primary hyperoxaluria	Martinez-Turrillas et al., 20227	CRISPR-SaCas9 knockout of Ldha; delivery by single AAV serotype 8	Inhibition of LDH, an enzyme (with AGT) involved in oxalate production in the liver	PH1 and PH3 mouse models	50%–60% indels in liver of treated PH1 mice; 40% in PH3 mice
Multiplex gene editing reduces oxalate production in primary hyperoxaluria type 1	Zheng et al. , 20238	CRISPR-Cpf1 delivered by single AAV with dual sgRNAs targeting Ldha and Hao1	Inhibition of Ldha and Hao1 enzymes	AgxtQ84X PH1 rats	Average indels of 14.1% and 25.1% at the Hao1 and Ldha loci, respectively
Efficient and safe therapeutic use of paired Cas9-nickases for primary hyperoxaluria type 1	Torella et al., 20249	Either dual AAV8 delivery of SaCas9 nickases + dual sgRNAs targeting exon 2 of mHao1 or an all-in-one AAV8 expressing these components	Disruption of mHao1	PH1 mice	57%–69% of Hao1 alleles edited in mice treated with dual AAVs; similar frequency (38.9%–58.7%) for all-in-one vector independent of dose
Efficient and safe in vivo treatment of primary hyperoxaluria type 1 via LNP-CRISPR-Cas9-mediated glycolate oxidase disruption	Jiang et al., 20243	LNP delivery of CRISPR-SpCas9 and sgRNA targeting either mAgxt, mHao1, or hHAO1	Inhibition of mHao1/hHAO1 enzymes	Systemic injection in WT mice of LNP-CRISPR targeting mAgxt; humanized hHAO1 knockin	<71% indels at Agxt (dose dependent); 61%–75% editing of mHao1; <59% editing at hHAO1	Optimization of dosing regimen, off-target

In clinical practice, recurrent stones caused by PH1 not only result in pain and renal dysfunction but also increase the economic burden of repeated surgeries, such as shockwave lithotripsy, percutaneous nephrolithotomy, or ureteroscopy. The Jiang et al. study introduces non-viral vectors, such as LNPs with low immunogenicity, offering new hope for PH1 patients. However, before CRISPR-based gene expression regulation systems can be applied clinically, several key issues must be addressed in future research: (1) Can CRISPR-based gene therapy prevent dialysis in PH1 patients and delay or reverse renal function decline? (2) Can this gene therapy be used for prenatal intervention to prevent PH1 development after birth? (3) While normal oxalate levels are beneficial for physiological functions, will long-term use of this gene therapy lower oxalate levels below normal and lead to potential side effects?

The research of Jiang et al. represents a valuable step forward, establishing a foundation for developing new CRISPR-Cas9-based non-viral vectors for PH1 treatment. In the future, the use of more efficient non-viral delivery vectors, such as cationic polymers,¹² along with the discovery of new target molecules, will further improve the therapeutic efficacy for PH1 (Figure 1).

Figure 1.

Current therapeutic strategies and future directions for treating primary hyperoxaluria type 1

Acknowledgments

This research was supported by the Cuiying Scientific and Technological Innovation Program of Lanzhou University Second Hospital (CY2021-MS-B03).

Declaration of interests

The authors declare no competing interests.