CRISPR Genome Editing Slows Triple-Negative Breast Cancer Growth

Significance 

Scientists at Boston Children’s Hospital and Northeastern University have developed a CRISPR genome editing system that suppresses the growth of triple-negative breast cancer (TNBC). A proof-of-principal study showed that a nanolipogel-delivered CRISPR system designed to target a known cancer gene halted tumor growth in a mouse model by 77%, without any evidence of toxicity to normal tissues. “Our results provide experimental evidence that in vivo CRISPR genome editing can halt TNBC tumor progression,” the team wrote in their published paper in the Proceedings of the National Academy of Sciences.

TNBC is the most aggressive of all the breast cancer types, and has the poorest prognosis. TNBC occurs more frequently in women under the age of 50 years, in African American women, and in those carrying the BRCA1 gene mutation. “Over 32,000 patients are estimated to be diagnosed with TNBC in the United States in 2019, representing 12% of all new breast cancer cases,” the team wrote. “The extremely aggressive and metastatic nature of TNBC, coupled with fewer treatment options, has resulted in the worst mortality rates among all breast cancer subtypes, highlighting an urgent and unmet clinical need for novel precision medicines to treat TNBC.”

While CRISPR genome editing shows promise as a potential treatment or even cure for genetic diseases including cancer, in practice, most clinically focused CRISPR studies have focused on straightforward monogenic diseases, which are caused by defects in a single gene. The therapeutic benefits of in vivo CRISPR genome editing in cancer is still unclear. No in vivo CRISPR genome editing has been investigated as a targeted therapeutic for TNBC.

Translating the potential of CRISPR genome editing into therapeutic platforms has been held back in part by the lack of effective delivery systems. Virally mediated delivery of the CRISPR elements isn’t suitable for carrying large payloads, and can potentially cause side effects in non-target cells. An alternative method involves encapsulating the CRISPR payload inside a cationic polymer or in lipid nanoparticles, but this can also result in toxicity, and the CRISPR elements may be trapped within the cell endosomes, or broken down before reaching the target cells.

The approach developed by the Boston Children’s Hospital team encapsulates the CRISPR system inside a soft, noncationic nanolipogel composed of nontoxic fatty molecules and hydrogels. The nanolipogel surface is then studded with antibodies targeting ICAM-1, a protein identified by the Moses laboratory in 2014 as a novel drug target for TNBC. Initial cell uptake studies indicated that the ICAM-1 antibodies effectively guide the CRISPR-containing nanolipogel particles to TNBC cells specifically. Further studies showed that, unlike stiffer nanoparticles, the soft tumor-targeted nanolipogel (tNLG) particles could readily fuse with the tumor cell membrane and deliver the CRISPR payload directly inside the cell.  tNLGs can selectively recognize and bind TNBC cells over normal breast cells, which may reduce their nonspecific toxicities in vivo, consistent with our previous findings using ICAM1 as a TNBC target,” the authors stated.  The deformable tNLGs can breach the tumor endothelial barrier more efficiently in vitro than their stiff counterpart, which may, in turn, enhance its in vivo performance.” As first author, Guo commented, “Using a soft particle allows us to penetrate the tumor better, without side effects, and with bigger cargo. Our system can substantially increase tumor delivery of CRISPR.”

To carry out proof-of-concept tests with the technology as a potential therapeutic strategy for TNBC, the researchers designed the CRISPR system to target Lcn2, an established cancer gene that they had previously found actively promoted breast cancer progression and metastasis, to generate tNLG-Lcn2KO nanolipogel particles. Tests in Lcn2-overexpressing human TNBC cell lines confirmed that the tNLG-Lcn2KO system was capable of “potent and efficient CRISPR genome editing,” and suppressed expression of Lcn2 at both the gene transcript and protein levels. CRISPR knockdown of Lcn2 in the TNBC cells led to reduced tumor cell aggressiveness and tendency to migrate, by inhibiting epithelial to mesenchymal transition, a mechanism that is implicated in promoting breast tumor progression and metastasis. The researchers say that even partial inhibition of EMT may “lead to a potent in vivo therapeutic benefit in TNBC therapy.”

They next injected the tNLG-Lcn2KO into TNBC-bearing mice, and found that within 24 hours of administration the ICAM-1-guided particles specifically homed in on the TNBC, at 1.7-fold higher levels than was achieved using control nanolipogel particles. The level of accumulation of tNLG-Lcn2KO in the mouse tumors was “significantly higher than the average tumor accumulation of conventional nanomedicines,” they noted.

To evaluate the therapeutic potential of the system TNBC-bearing mice received weekly injections of the tNLG-Lcn2KO nanolipogel particles, for four weeks, and the tumors were allowed to continue to grow for another four weeks. The results showed that compared with control animals, tNLG-Lcn2KO treatment reduced tumor volume by 77%, and tumor weight by 69%. The in vivo editing efficiency was about 81%, resulting in significantly reduced lcn2 protein levels in the tNLG-Lcn2KO treatment group compared with the results in mice receiving sham treatment. “These in vivo results provide experimental evidence that efficient in vivo CRISPR genome editing by tNLGs can generate a potent and specific therapeutic benefit against TNBC tumor growth.”

Encouragingly, there was no evidence of treatment-related toxicity in organs including the liver, spleen, and kidney, indicating that “the nanoformulation of tNLG is relatively safe to use for in vivo CRISPR genome editing.”

The research team concluded that their proof-of-principle study suggests that the tNLG formulation has “a promising and broad potential for translating CRISPR genome editing into a novel precision medicine in cancer therapy.” It should also be feasible to use tNLG as a platform delivery system to target TNBC using other targets to guide the nanolipogel particles to the TNBC tumors in vivo, they suggested. And different TNBC oncogenes, such as PIK3CA, WNT, and Notch, could also serve as alternative genome editing targets for Lcn2-negative TNBC subtypes, they pointed out. Their system can deliver significantly more drug to the tumor, in a precise and safe way.

CRISPR Genome Editing Holds Back Triple-Negative Breast Cancer in Mice - Medicine Innovates

About the author

Dr. Marsha A. Moses is the Julia Dyckman Andrus Professor at Harvard Medical School and the Director of the Vascular Biology Program at Boston Children’s Hospital. She has made significant contributions to our understanding of the biochemical and molecular mechanisms that underlie the regulation of tumor growth and progression.

Dr. Moses and her laboratory have discovered a number of inhibitors of neovascularization that function at both the transcriptional and translational level some of which are being developed for potential clinical use in a variety of human diseases. Named a pioneer in the field of Biomarker Medicine by the Journal of the National Cancer Institute, she created a Proteomics Initiative at Boston Children’s Hospital and has utilized its resources, including an extensive human biorepository and her significant expertise in proteomics, to discover and validate a number of novel, non-invasive, urinary biomarkers for a variety of human cancers as well as non-neoplastic diseases.

A number of these biomarkers are being used in clinical trials. She and her team have also engineered novel non-toxic, targeted nanomedicines for the treatment of human cancers and their metastases. These drug delivery systems are designed to deliver a variety of therapeutic agents including siRNAs, chemotherapies and gene editing systems. A number of these diagnostics and potential therapeutics are included in Dr. Moses’ significant patent portfolio composed of both US and foreign patents.

Dr. Moses’ basic and translational work has been published in such journals as Science, The New England Journal of Medicine, Cell, and Nature Communications, among others. Marsha received a Ph.D. in Biochemistry from Boston University and completed a National Institutes of Health postdoctoral fellowship at Boston Children’s Hospital and MIT in the laboratory of Dr. Robert Langer. She is the recipient of a number of NIH and foundation grants and awards.

Dr. Moses has been recognized with both of Harvard Medical School’s mentoring awards, the A. Clifford Barger Mentoring Award (2003) and the Joseph B. Martin Dean’s Leadership Award for the Advancement of Women Faculty (2009). In 2014, she received the Excellence in Mentoring Award from the Postdoc Association of Boston Children’s Hospital and in 2016, she received their award for Exceptional Mentorship. In 2013, Dr. Moses received the Honorary Member Mentoring Award from the Association of Women Surgeons of the American College of Surgeons.

Dr. Moses was elected to the Institute of Medicine (National Academy of Medicine) of the National Academies of the United States in 2008, the National Academy of Inventors in 2013 and the American Institute for Molecular and Biological Engineering in 2018.

Reference

Peng Guo, Jiang Yang, Jing Huang, Debra T. Auguste, and Marsha A. Moses. Therapeutic genome editing of triple-negative breast tumors using a noncationic and deformable nanolipogel. PNAS September 10, 2019 116 (37) 18295-18303

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