Disrupting the splice site instead of the mutation itself extended lifespan 2.4-fold in progeria mice and restored breeding in previously infertile pairs
Disrupting the splice site instead of the mutation itself extended lifespan 2.4-fold in progeria mice and restored breeding in previously infertile pairs
On September 18, a preprint from Zhongjun Zhou's laboratory (University of Hong Kong) appeared on bioRxiv. Rather than correcting the mutation in the LMNA gene directly, the authors used CRISPR-Cas9 to destroy the splice site, the signal the cell uses to cut and rejoin a stretch of RNA and which this mutation merely strengthens. A single injection in newborn mice raised median survival from 115 to 279 days and restored breeding in previously infertile pairs.
In Hutchinson-Gilford progeria syndrome, a childhood progeroid disease, a single-letter substitution in the LMNA gene strengthens a cryptic splice site that is barely used under normal conditions, rather than damaging lamin A itself, the structural protein of the nuclear envelope. As a result, part of the RNA is mis-spliced: instead of lamin A, the cell produces a truncated, toxic protein called progerin, which accumulates at the nuclear envelope and triggers premature aging of blood vessels, bone, and skin.
A similar strategy was attempted in 2011, when the aberrant site was silenced with antisense oligonucleotides, but that approach required injections for life. The new work moves the same target to the DNA level and makes the intervention a one-time event. The mutation sits only six nucleotides from the conserved GT dinucleotide, the anchor the cell uses to locate the splice site. The CRISPR guide RNA binds preferentially to the disease allele and cuts the DNA at this anchor rather than in the region shared by progerin and healthy lamin A. Any break that destroys the splice donor counts as a success. This is gentler than the single-letter precision required by earlier-generation editors and more specific than previous CRISPR approaches, which cut the shared coding sequence and slightly reduced healthy protein as well.
"An activating mutation does not have to be corrected precisely; the splice signal that makes it pathogenic can be disabled instead,"the authors write.
In patient fibroblasts, this suppressed progerin production by 92.7–96.8% while preserving healthy lamins A and C. A genome-wide off-target search found no unintended cuts in coding genes.
A single injection of AAV9 viral vector in newborn mice raised median survival from 115 to 279 days, an increase of 143% (33 mice in the control group, 52 treated). This is several times greater than the 26–27% gain from earlier CRISPR disruption of the same gene and the 22–30% gain from suppression of the innate immune signaling protein STING with the antagonist H-151 in the same model. The effect was systemic: smooth muscle cell counts in the aortic wall rose to 1.4–1.9 times the control level, cardiac, hepatic, and skeletal muscle fibrosis fell by 65–86%, and bone density increased.
The most unexpected result was the restoration of fertility. Before this study, no pair of mice homozygous for the progeria mutation had ever produced offspring. After treatment, every treated pair bred successfully, yielding 52 pups across seven litters. Ovarian follicles were preserved in females, and seminiferous tissue in the testes thickened in males.
"To our knowledge, this is the first reported case of offspring from such pairs following an intervention administered in the first days of life,"the authors note.
The experimental mice already carried a Cas9 transgene, so the virus delivered only the guide RNA. In humans, both components would need to be delivered together, and compact Cas9 variants suitable for this purpose already exist.
The principle extends beyond a single disease. Similar cryptic splice sites underlie a subset of cystic fibrosis cases and one form of therapy-resistant prostate cancer. Progeria is used as a model of accelerated aging, and the systemic restoration seen in these mice positions regulatory-signal disruption as a standalone tool in gene therapy for aging.