Bowdoin Science Journal

CRISPR

Engineered Nanoparticles Enable Selective Gene Therapy in Brain Tumors

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Inborn protective mechanisms present challenges for therapies targeting cancers of the brain. Engineered nanoparticles permit the selective delivery of CRISPR-Cas9 to glioblastoma tumors.

Glioblastoma accounts for almost half of all cancerous tumors originating in the brain.1 Even with maximum safe treatment, the median survival period for patients is less than 1.5 years.2 However, survival varies widely by age, from a 0.9% 5-year survival rate for patients over 75 years old to an 18.2% 5-year survival rate for patient 0-19 years old.3 Nevertheless, the overall 5-year survival rate remains low at 5%2 and novel therapies are urgently needed for glioblastoma treatment. Zou et al. present a highly specific CRISPR-Cas9-based gene therapy for glioblastoma.4

Gene therapies offer an attractive treatment for cancers. The goal of gene therapy is to mutate or remove deleterious DNA sequences such that they are unable to be transcribed and translated into functioning proteins. In the most prevalent technique, CRISPR-Cas9, single guide RNA (sgRNA) identifies and binds to the target DNA sequence. It then recruits Cas9 proteins to excise parts of the target DNA sequence. Mutations are generated as the cell tries to repair the damaged DNA.5

While a powerful tool, researchers have struggled to effectively deliver CRISPR-Cas9 to their cellular target. Delivery is particularly complex in brain tumors such as glioblastoma. Traditionally medications are trafficked through the body and delivered to their target through the bloodstream. However, the brain is a more complex and protected system. Primarily, selective delivery of therapeutics to tumor cells is necessary to protect neuronal function. Moreover, the brain is separated from the blood stream by a thin layer of cells termed the blood-brain barrier (BBB). The BBB allows for selective permeation of compounds into the brain, shielding neurons from toxins while permitting the passage of essential nutrients.6 Previous studies have sought to transport CRISPR-Cas9 therapies across the BBB using viruses as delivery capsules7 or circumvent the BBB altogether via intercranial injection of therapeutics.8 Yet these methods carry risk, either an immune response to the viral vector or complications from the invasive injection.

Zou et al. sought to resolve the issues of specific cell targeting and BBB permeability in CRISPR-Cas9 delivery by encapsulating the gene editing complex within a nanoparticle. The researchers chose to target Polo-like kinase 1 (PLK1) using CRISPR-Cas9 gene therapy. PLK1 is an attractive for selective gene therapy due to its higher overexpression by glioblastoma cells and by the proliferative glioblastoma subtype in particular.9 Moreover, inhibition of PLK1 has been shown to reduce tumor growth and induce cell death.9

The researchers encapsulated Cas9 and sgPLK1 in a neutrally charged nanoparticle for delivery. The small size of nanoparticles, which are measured in nanometers, allow for easy transport through the bloodstream and uptake by cells. Taking advantage of the high expression of lipoprotein receptor-related protein-1 (LRP-1) on both BBB endothelial cells and glioblastoma tumor cells, they decorated the nanocapsule surface with LRP-1 ligand angiopep-2 peptide to facilitate selective uptake by BBB and glioblastoma cells (Figure 1). Zou and her colleagues bound the nanoparticle together with disulfide bonds as an added layer of selectivity for glioblastoma cell delivery. In the high glutathione environment of a glioblastoma cell, the nanoparticle dissolves, releasing its contents. However, glutathione concentrations are lower in BBB endothelial cells and healthy neurons, reducing the dissolution of the nanoparticles and leaving healthy DNA alone.

 

Figure 1. Nanoparticles enable permeation of the blood brain barrier (BBB) and selective delivery of the CRISPR/Cas9 system to glioblastoma (GBM) cells. Angiopep-2 peptides, which decorate the nanoparticle’s surface, bind with lipoprotein receptor-related protein-1 (LRP-1) overexpressed on BBB and GBM cells. Following uptake into GBM cells, the nanoparticles dissolve in the high glutathione environment, releasing the Cas9 nuclease and single guide RNA (sgRNA) targeting Polo-like kinase 1 (PLK-1) genes. Zou et al. demonstrated the selective mutation of PLK-1 induced apoptosis in GBM cells with minimal off-target effects.

 

Through Cas9/sgPLK1 delivery by nanoparticle, Zou et al. demonstrated a 53% reduction in expression of the targeted gene in vitro. PLK1 gene editing was cell selective, with negligible genetic mutation in the healthy surrounding brain tissue. While nanoparticles with and without disulfide cross-linking were capable of gene editing, the disulfide cross-linked nanoparticle induced almost 400% more mutations. Glioblastoma cells treated with disulfide cross-linked nanoparticles were also over 3 times more likely to undergo apoptosis cell death. Mice grafted with patient glioblastoma tumors treated with Cas9/sgPLK1 nanocapsules experienced an almost 3-fold extension in life expectancy, suggesting this treatment as a viable anti-glioblastoma therapy.5

Yet in order to be effectively applied as a cancer therapy, the efficiency of this nanoparticle delivery system must be increased. In mouse glioblastoma models, Zou et al. only achieved a maximum accumulation of 12% and effected a 38% knockdown of PLK1.5 Despite their low magnitude, these values are far greater than similar gene therapy treatments currently studied, suggesting nanoparticles present an innovation in the delivery of CRISPR-Cas9 to difficult to access tumors.

In addition to the improved efficacy over existing systems, the work of Zou et al. opens the door for less invasive administration of treatment. In contrast to previous gene therapies administered directly to the tumor through intercranial injection, the BBB penetration and tumor-specific accumulation of the nanoparticles may permit systemic administration. Zou et al. injected the nanoparticles intravenously, but treatment may even be given as a pill taken orally, eliminating any surgical intervention. Moreover, due to their modular design, the engineered nanoparticles may be adapted as targeted treatments for other tumors. Exchanging the angiopep-2 peptides for another ligand would facilitate uptake by cells expressing the corresponding receptor. The load carried within the nanoparticle could be altered to contain a different sgRNA targeting a new gene or another therapeutic entirely as a complementary treatment. Nanoparticle delivery systems like that studied by Zou et al. contains many layers of selectivity, offering hope for effective delivery of treatment to previously inaccessible tumors.

 

Works Cited:

  1. Wirsching, H.-G. & Weller, M. Glioblastoma. in Malignant Brain Tumors : State-of-the-Art Treatment (eds. Moliterno Gunel, J., Piepmeier, J. M. & Baehring, J. M.) 265–288 (Springer International Publishing, Cham, 2017). doi:10.1007/978-3-319-49864-5_18.
  2. Delgado-López, P. D. & Corrales-García, E. M. Survival in glioblastoma: a review on the impact of treatment modalities. Clin Transl Oncol 18, 1062–1071 (2016).
  3. Ostrom, Q. T. et al. CBTRUS Statistical Report: Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2007–2011. Neuro Oncol 16, iv1–iv63 (2014).
  4. Lino, C. A., Harper, J. C., Carney, J. P. & Timlin, J. A. Delivering CRISPR: a review of the challenges and approaches. Drug Delivery 25, 1234–1257 (2018).
  5. Zou, Y. et al. Blood-brain barrier–penetrating single CRISPR-Cas9 nanocapsules for effective and safe glioblastoma gene therapy. Sci Adv 8, eabm8011.
  6. Dotiwala, A. K., McCausland, C. & Samra, N. S. Anatomy, Head and Neck: Blood Brain Barrier. in StatPearls (StatPearls Publishing, Treasure Island (FL), 2024).
  7. Song, R. et al. Selection of rAAV vectors that cross the human blood-brain barrier and target the central nervous system using a transwell model. Molecular Therapy Methods & Clinical Development 27, 73–88 (2022).
  8. Lee, B. et al. Nanoparticle delivery of CRISPR into the brain rescues a mouse model of fragile X syndrome from exaggerated repetitive behaviours. Nat Biomed Eng 2, 497–507 (2018).
  9. Lee, C. et al. Polo-Like Kinase 1 Inhibition Kills Glioblastoma Multiforme Brain Tumor Cells in Part Through Loss of SOX2 and Delays Tumor Progression in Mice. Stem Cells 30, 1064–1075 (2012).

CRISPR-izing the Mitochondrial Genome Is Here

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CRISPR-Cas9 has become a standard method for editing the nuclear genome, but researchers have long debated whether the mitochondrial genome could be edited too. At last, we have an answer: yes.

When you hear the word “genome,” what comes to mind? The collection of DNA present in the nucleus? While technically correct, our old friend, the “powerhouse of the cell,” would like to object: it too has a genome! In humans, the mitochondrial genome is a circularized strand of DNA that is around 16,500 base pairs long, and within a single cell, it can have hundreds to thousands of copies [1]. At first glance, mitochondrial DNA (mtDNA) doesn’t seem that impressive, especially compared to the 3.4 billion base pairs of the nuclear genome. However, it has significant relevance to human health: mtDNA mutations are associated with numerous diseases from the cardiomyopathy of Barth syndrome to the neurodegeneration of Leigh syndrome [2]. Thus, efforts have been made to “fix” these defects, particularly through CRISPR-Cas9 gene editing.

The CRISPR-Cas9 system consists of an enzyme (Cas9) and a guide RNA (sgRNA) that can locate a specific region of the genome and induce a double-stranded break in the DNA. Using DNA repair mechanisms, this can be exploited to delete or insert a new sequence. For instance, homology-directed repair (HDR) uses a DNA template, either natural or supplied by researchers, to rebuild the broken strand with that template’s sequence. This has been especially powerful for editing nuclear genomes, but it has been debated whether this method could be used for mtDNA as well [3].

This is where Bi et al. enter the scene with their recent study [4]. Their goal was simple: to demonstrate that the mitochondrial genome can, in fact, be “CRISPR-ized.” The first step was to design a CRISPR-Cas9 system capable of entering the mitochondrion. To accomplish this, they took an existing system for nuclear editing and inserted “localization signals” that tell the cell where the protein needs to go. One of these was the mitochondrial targeting signal (MTS) derived from a nuclear-encoded gene, COX8A, that allows the protein to travel to the mitochondrion. Next, an sgRNA was designed that targets the ND4 gene of the mitochondrial genome. Combining these, Bi et al. created a system that could be tested in human cells. Indeed, with the use of fluorescent markers and microscopy, they demonstrated that both the Cas9 protein and sgRNA could enter the mitochondrion (Figure 1).

Next, the researchers needed to determine whether this system could edit mtDNA once inside the organelle. In addressing this, they added the CRISPR-Cas9 system alongside a template strand (called an ssODN) that contained an inserted sequence, GAATTC, that they could screen for (Figure 1). Once the cells were treated, they confirmed the insertion through two methods: PCR and direct sequencing. The PCR method used a primer that could only bind to the inserted sequence, so if the mtDNA was amplified, that indicated a successful “knockin.” The sequencing method, “PCR-free 3rd generation sequencing,” provided additional confirmation without the risk of false-positives from PCR artifacts (Figure 1).

Finally, as a proof of concept, Bi et al. altered the sgRNA and template strand to insert a pathogenic mtDNA mutation (changing ACCTTGC to GCAAGGT on the ND1 gene). This is associated with mitochondrial myopathy (a disease that causes muscular problems), and if successfully inserted, it could provide researchers with a new cell that models this mutant. Indeed, PCR and sequencing confirmed its knockin, demonstrating the power of editing mtDNA.

The authors of this paper directly confirmed that mtDNA could be edited via CRISPR-Cas9 for the first time. This is in contrast to previous studies which have shown mitochondrial localization of the Cas9-sgRNA complex, but with no clear indication of knockin because of PCR-based artifacts or indirect screening [5]. However, the present study is not without its own limitations, particularly in the method’s feasibility at a clinical level. First and foremost, an individual cell has many more copies of mtDNA than nuclear DNA, so editing every strand is more difficult. Indeed, the “knockin efficiency,” or proportion of DNA with the GAATTC insertion, was only 0.03-0.05% (compared to the 20-30% seen in some nuclear genome editing studies) [6]. Furthermore, Bi et al. reported an increase of reactive oxygen species (ROS) formation upon the addition of CRISPR-Cas9, a class of reactive molecules that can degrade DNA and induce cell death. 

Work remains to be done, whether it is through improving the efficacy of the CRISPR-CAS9 system or confirming the method’s safety amidst ROS production. Nonetheless, Bi et al. give us a powerful starting point for treating mitochondrial dysfunction in ways never previously thought possible. In the coming years, CRISPR-izing mtDNA may even become a standard technique, just as we saw with nuclear DNA in the last decade.

References:

  1. Falkenberg M, Larsson NG, Gustafsson CM. (2007). DNA replication and transcription in mammalian mitochondria. Annu Rev Biochem, 76: 679-99. https://doi.org/10.1146/annurev.biochem.76.060305.152028. 
  2. Stenton SL, Prokisch H. (2020). Genetics of mitochondrial diseases: Identifying mutations to help diagnosis. EBioMedicine, 56: 102784. https://doi.org/10.1016/j.ebiom.2020.102784.
  3. Gammage PA, Moraes CT, Minczuk M. (2018). Mitochondrial Genome Engineering: The Revolution May Not Be CRISPR-Ized. Trends Genet, 34(2): 101-110. https://doi.org/10.1016/j.tig.2017.11.001. 
  4. Bi R et al. (2022). Direct evidence of CRISPR-Cas9-mediated mitochondrial genome editing. Innovation (Camb), 3(6): 100329. https://doi.org/10.1016/j.xinn.2022.100329. 
  5. Antón Z et al. (2020). Mitochondrial import, health and mtDNA copy number variability seen when using type II and type V CRISPR effectors. J Cell Sci, 133(18): jcs248468. https://doi.org/10.1242/jcs.248468. 
  6. Zhang JP et al. (2017). Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage. Genome Biol, 18(1): 35. https://doi.org/10.1186/s13059-017-1164-8.