CRISPR Therapy Reduces Influenza A Viral Load by 50% in Human Lung Chip Model
核心洞察
Harvard researchers developed a novel CRISPR (搜索)-based therapy that reduced Influenza A virus (搜索) load by more than 50% in human lung chip models after a single treatment.
The therapy targets conserved sequences in the viral PB1 gene and demonstrated minimal off-target effects while significantly reducing inflammatory responses.
The human lung alveolus chip platform enables clinically relevant testing of CRISPR (搜索) RNA therapies, overcoming limitations of animal models for human-specific treatments.
Researchers at Harvard University's Wyss Institute for Biologically Inspired Engineering have demonstrated that a novel CRISPR (搜索)-based therapy can reduce Influenza A virus (搜索) (IAV (搜索)) load by more than 50% in human lung tissue models, marking a significant advance in the development of pan-influenza treatments that could overcome the virus's notorious ability to develop resistance.
The breakthrough study, published in Lab on a Chip, utilized a microfluidic "breathing" human lung alveolus chip to test CRISPR (搜索) machinery targeting conserved sequences in IAV (搜索)'s genome. The research addresses a critical challenge in influenza treatment development: IAV's capacity to mutate, rearrange genetic information, and recombine with other viruses, making it extremely difficult to develop effective antivirals.
Novel CRISPR Design Targets Viral Weak Points
The research team, led by Founding Director Donald Ingber, M.D., Ph.D., designed CRISPR (搜索) machinery using two CRISPR RNAs (crRNAs (搜索)) that target invariable regions in the polymerase basic 1 (搜索) (PB1) gene of IAV (搜索). These sequences are conserved across the vast majority of IAV viruses that infect humans, making them ideal targets for a pan-influenza approach.
"Our findings demonstrate that the human Lung Chip model of IAV (搜索) infection is a highly valuable preclinical testbed for CRISPR (搜索) RNA therapeutics that act broadly across virus strains," said Ingber, who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children's Hospital.
When researchers introduced the crRNAs (搜索) along with an mRNA molecule encoding the RNA-destroying Cas13 (搜索) enzyme, the system achieved more than 80% reduction of PB1 RNA levels in cultured lung cells. The crRNAs guided Cas13 to the PB1 target sequence of different IAVs used to infect the cells.
Advanced Drug Delivery Platform
A critical component of the study's success was the development of an effective delivery system. The team, working with Associate Director Natalie Artzi, Ph.D., packaged the CRISPR (搜索) machinery in polymer-based nanoparticles with specific affinity to lung epithelial cells.
"Delivery remains the key bottleneck for nucleic acid therapeutics. Our work on delivering CRISPR (搜索)-based antivirals shows that nanoparticle formulation critically determines efficacy, particularly in primary human alveolar cells," said Artzi, who is also the Hansjörg Wyss Associate Professor of Biologically Inspired Engineering at Harvard Medical School and Brigham and Women's Hospital.
The nanoparticle delivery system enabled efficient packaging of therapeutic RNA molecules and their internalization by human lung alveolar tissue, a challenge that had previously limited CRISPR (搜索) antiviral development.
Human Lung Chip Enables Clinically Relevant Testing
The study's foundation was the human Lung Chip platform, which mimics the lung's tiny air sacs (alveoli) where IAV (搜索) infection occurs. The chip contains two parallel channels: one with human primary alveolar epithelium and another with human pulmonary vascular endothelial cells, separated by a porous membrane that allows molecular and gas exchange.
The system can simulate natural blood flow and breathing patterns by flowing culture medium through the vascular channel and circulating air through the epithelial channel while cyclically stretching the tissue. This creates a physiologically relevant model that faithfully replicates IAV (搜索) infection dynamics, including virus entry, replication, and inflammatory responses.
First author Yuncheng Man, Ph.D., a postdoctoral fellow in Ingber's lab, noted the platform's unique capabilities: "For the first time, we were able to assess potential off-target effects of an IAV (搜索)-specific CRISPR (搜索) therapy in living human lung tissues."
Minimal Off-Target Effects Observed
Safety assessment revealed encouraging results regarding potential off-target effects. Transcriptomic analysis showed that the non-targeting control CRISPR (搜索) system did not affect gene expression in the Lung Chips. The genes that were changed by the targeted pan-IAV (搜索) version did not share sequence identities with the targeted IAV sequence but were associated with downregulated host inflammatory pathways.
"This strongly argues for the general safety of our approach," said Man, emphasizing that the system enables assessment of both efficacy and safety in a clinically relevant human tissue model.
Addressing a Global Health Challenge
The research addresses a significant public health burden. IAV (搜索) has caused six major flu pandemics responsible for 50 to 100 million deaths globally. In the United States alone, despite seasonally updated vaccines, IAV infections lead to an estimated 140,000 to 710,000 hospitalizations and 12,000 to 52,000 deaths annually.
The study tested the system using various IAV (搜索) strains, including the pandemic H3N2 strain that caused a global flu pandemic with 1 million deaths in 1968. The platform successfully replicated the intricacies of IAV infection and demonstrated the therapeutic potential of the CRISPR (搜索) approach.
Overcoming Traditional Model Limitations
Previous efforts to develop IAV (搜索) antivirals have relied heavily on animal models including mice, hamsters, and ferrets. However, these models have significant limitations: they differ from humans in anatomy, physiology, and genetic design, affecting virus entry and replication efficiency. Their immune systems also function differently than humans, and drug delivery requirements differ substantially.
The human-specific nature of CRISPR (搜索) targets has made animal model studies particularly challenging, as the sequences being targeted are so human-specific that meaningful studies cannot be conducted in animal models.
The research was supported by the Defense Advanced Research Projects Agency (DARPA (搜索)) under Cooperative Agreement HR0011-22-2-0017 and the Wyss Institute (搜索) at Harvard University. The collaborative effort included researchers Ryan Posey, Haiquing Bai, Amanda Jiang, Pere Dosta, Diana Ocampo-Alvarado, Robert Plebani, Jie Ji, and Chaitra Belgur.
This advancement represents a significant step toward developing effective treatments that could help prevent future influenza pandemics by targeting conserved viral sequences that are less likely to mutate, potentially saving thousands of lives.