Technical Review: The First CRISPR-Based Therapeutic (SL_1.52) for African Swine Fever Is Effective in Swine

Authors: Naveen Verma, Alison O’Mahony, Roky Mohammad, Dylan Keiser, Craig W. Mosman, Deric Holden, Kristin Starr, Jared Bauer, Bradley Bauer, Roypim Suntisukwattana, Waranya Atthaapa, Angkana Tantituvanont, Dachrit Nilubol, and Douglas P. Gladue​

Affiliations: Seek Labs, Salt Lake City, Utah, USA

Background Introduction

African swine fever virus (ASFV) is a large, structurally complex double-stranded DNA virus with a 180–190 kbp genome encoding over 150 genes. It causes African swine fever (ASF) with mortality rates reaching 90–100% and has become a major threat to global pig production. The virus spread from sub-Saharan Africa to Eurasia after 2007 and was detected in the Dominican Republic and Haiti in 2021, marking the emergence of the Eurasian pandemic genotype in the Western Hemisphere. Current disease control relies on diagnostics, movement restrictions, and mass culling, all of which have proven insufficient to contain ongoing pandemics.

Currently available live-attenuated vaccines use recombinant viruses subjected to gene deletions. More recently however, CRISPR/Cas systems have undergone extensive research as a promising alternative. Their programmability via guide RNAs (gRNAs) makes them more readily adjustable, needing only sequence information rather than detailed protein or epitope knowledge. Clinically, Cas9 therapies have been primarily researched in humans, with treatments such as Casgevy approved for sickle cell disease and beta-thalassemia. This study reports the first in vivo therapeutic application of a CRISPR/Cas9 system (SL_1.52) against ASFV in pigs, targeting the highly conserved DNA polymerase gene G1211R in pandemic biotype 2 and hybrid 1/2 strains.

Materials and Methodology

The researchers first conducted gRNA and plasmid design to target G1211R, the open reading frame (ORF) encoding ASFV DNA polymerase. Cas9 ribonucleoprotein (RNP) complexes of sgRNA were tested to verify targeting performance in vitro, first administered directly and then as a SL_1.52 plasmid encoding the RNP and gRNAs. This plasmid was then encapsulated in lipid nanoparticles (LNPs) for further in vivo experiments, where pigs were challenged with ASFV after vaccination.

Results

CRISPR Design and Biochemical Validation

The two gRNAs in SL_1.52 demonstrate specific and efficient cleavage of the G1211R target sequence in biochemical assays. RNPs containing Cas9 and either gRNA1 or gRNA2 independently converted supercoiled G1211R plasmid to linear form, consistent with single cleavage events at each target site. When both gRNAs were used together, two DNA fragments of approximately 1651 bp and 7747 bp were produced, matching the predicted dual cleavage pattern between the two target sites. This functional validation confirms that both guides are competent and supports the dual-guide design strategy to minimize the probability of escape mutations through single-site alterations.

Cell-Based Target Knockdown in HEK293T Cells

In mammalian HEK293T cells, SL_1.52 exhibits high specificity and reduces expression of the targeted G1211R gene but not the non-targeted P1192R control gene. Co-transfection of SL_1.52 with the G1211R-targeting plasmid resulted in a statistically significant reduction in G1211R transcript levels compared with control conditions (p ≤ 0.01), whereas co-transfection with the P1192R non-target plasmid showed no significant changes in P1192R expression. These results demonstrate functional CRISPR activity in a mammalian cellular context and confirm that SL_1.52 acts as a targeted antiviral nuclease rather than a broadly mutagenic agent.​

The relative gene expression of G1211R (targeted) and P1192R (non-targeted) when transfected with or without SL_1.52 was determined by qPCR for the indicated gene and relative gene expression was determined in the absence or presence of SL_1.52, graphed in GraphPad Prism (Version 10.2.1), and statistical significance was determined by non-parametric Student’s t test. ** = p-value ≤ 0.01; ns = non-significant.

In Vivo Therapeutic Efficacy

SL_1.52 treatment substantially reduces viral load and improves survival in pigs challenged with a lethal ASFV dose. qPCR analysis of blood revealed significantly lower viral loads in SL_1.52-treated pigs at 10 days post-infection compared to untreated controls (Mann–Whitney U test with BKY FDR correction, q = 0.0018). Four of seven treated pigs (SL_1.52-8, -10, -11, -12) survived beyond all untreated animals and achieved complete clearance of detectable viremia in blood by day 35 post-infection, representing a 57% survival rate in an otherwise uniformly lethal infection model. In contrast, all seven control pigs succumbed to acute ASFV between days 10 and 25 post-infection. Clinical observations revealed that surviving treated animals remained asymptomatic after initial disease onset, whereas untreated animals developed progressive and severe ASF manifestations including depression, anorexia, weakness, and cutaneous hemorrhages, underscoring the therapeutic efficacy of SL_1.52 in ameliorating disease progression and enabling viral clearance.

Overall survival of animals infected with a lethal dose of ASFV followed by the SL_1.52 treatment compared to control animals over a period of 35 days post treatment. Seven swine in each group were inoculated intramuscularly (IM) with 102 HAD50 of ASFV/Thailand/2023, and treatment was administered at 5 dpi and 6 dpi.

Surviving SL_1.52-treated pigs developed robust anti-ASFV antibody responses and demonstrated durable protective immunity against homologous re-challenge. ASFV-specific antibody levels were near-background at baseline (−1 dpi) in all animals but rose significantly by day 10 and exceeded the 40% positive threshold by day 30 post-infection in all infected animals. Critically, when all surviving treated pigs and naïve control pigs were re-challenged at day 62 with a second lethal dose of ASFV/Thailand/2024, all four previously surviving SL_1.52-treated pigs remained resistant to disease, whereas all naïve control animals succumbed by day 12 post-challenge. These findings demonstrate that SL_1.52 enables sufficient antigen presentation and immune recognition to establish protective, durable immunity against homologous viral re-exposure, functioning as a therapeutic rescue that both clears acute infection and generates adaptive immune memory.

Conclusion

Verma and colleagues established SL_1.52 as the first demonstrated CRISPR/Cas9-based therapeutic effective against African swine fever virus in live swine. Plasmid DNA-LNP–delivered CRISPR can significantly reduce viral load, rescue a substantial fraction of pigs from a lethal ASFV challenge, drive viral clearance, and enable protective immunity against homologous re-challenge. Altered gRNA sequences may be used to retarget emerging ASFV strains or additional viral loci, while the LNP carriers could also be optimized for enhanced tissue targeting.

This paper utilized the PreciGenome NanoGenerator Flex-M for LNP synthesis. Uniform synthesis conditions are essential for consistent encapsulation of DNA in LNPs, while pressure-based microfluidics are highly scalable for pre-clinical experiments on animals of various sizes.

For further details, check out the link to the article here:

https://pmc.ncbi.nlm.nih.gov/articles/PMC12656968/