CRISPR/CAS9 Therapeutics Development

CRISPR/Cas9 technology is the latest edition of gene-editing tools that involved both guide RNA and CAS nuclease engineering. This technology offers the potential to restore any genetic disease of human in fast action, cost-effective and have a better accuracy than previous gene editing techniques with a broad range of applications in basic and medical research. Seattle Genova offers a wide range of products and services about CRISPR/Cas9 to develop therapeutic cell services for our clients.

Background of CRISPR/Cas9

Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated protein (CAS) system is first developed in 2012. In the first five years, the application has been tested in different cell types, improving its efficiency at cutting DNA and then clinical application which reported promising results. The CRISPR/Cas9 system is among adaptive immune systems that found in bacteria and archaea, in which provides immunity against bacteriophages, the viruses that attack bacteria.

CRISPR/Cas9 technology has emerged to a broad range of applications across multiple fields, including as a treatment for human diseases. In simple words, CRISPR/Cas9 could let us edit any genetic mutation at will to treat any gene-related disease. However, it is still in the early stages of therapeutic development.

Here is a list of diseases that we, Seattle Genova could custom for you:

1.       Chronic urinary tract infection (UTI)

A chronic UTI is one of a bacterial infection that enter the urinary system through the urethra, and then they grow in the bladder for a long period even had prescribed with antibiotics. UTI cause a burning sensation during urination and the need to urinate frequently. It’s helpful to break down UTIs into bladder and urethral infections to better understand how they develop.

Regarding the current treatment with the application of CRISPR/Cas9 system, there is a clinical trial that combines Cas9 with Cas3 in the bactericidal activity. Three bacteriophages combined with CRISPR-Cas3, designed to attack the genome of the three strains of E. coli that kills it. Bacteriophages usually work by injecting their genetic material into bacteria and using the bacteria as a factory to make more bacteriophages. Eventually, the bacteria will burst, dying as they release more copies of the phage.

In this treatment, phages have been engineered to be an even more powerful tool against E. coli. In addition to the natural action of phages that kills bacteria, these bacteriophages contain CRISPR-Cas3 in their genome. While the more-famous Cas protein Cas9 makes a precise cut at a single location, Cas3 shreds DNA at the gene regions it is targeted to find. In this treatment, the CRISPR-Cas3 system is made to target the genomes of the targeted E. coli strains and damage them by shredding stretches of DNA. In experiments on isolated cells and in animals with urinary tract and other infections, the addition of CRISPR-Cas3 makes phages much more effective at killing E. coli.

2.       Blood disorder

The blood disorders beta-thalassemia and sickle cell disease, which affect oxygen transport in the blood, are the target of a CRISPR/Cas9 treatment. Both diseases are caused by mutations in the hemoglobin β subunit gene (HBB). Mutations in HBB that cause TDT4 result in reduced (β+) or absent (β0) β-globin synthesis and an imbalance between the α-like and β-like globin (e.g., β, γ, and δ) chains of hemoglobin, which causes ineffective erythropoiesis (Frangoul et al, 2021).

The therapy consists of harvesting bone marrow stem cells from the patients and using CRISPR/Cas9 technology in vitro to make them produce fetal hemoglobin. This is a natural form of the oxygen-carrying protein that binds oxygen much better than the conventional adult form. The modified cells are then reinfused into the patient.

3.       Cancer

A study in China was testing the use of CRISPR to modify immune T cells extracted from the patient. The gene-editing technology is used to remove the gene that encodes for a protein called PD-1. This protein found on the surface of immune cells is the target of some cancer drugs such as checkpoint inhibitors. This is because some tumor cells are able to bind to the PD-1 protein to block the immune response against cancer. The trial tested this approach in 12 patients with non-small cell lung cancer at the West China Hospital. The results, published in April 2020, suggested the approach was feasible and safe.

Another research group at Duke University Medical Center, North Carolina, USA has used a similar approach in cervical carcinoma to demonstrate that expression of a bacterial Cas9 RNA-guided endonuclease, together with single guide RNAs (sgRNAs) specific for E6 or E7, is able to induce cleavage of the HPV genome, resulting in the introduction of inactivating deletion and insertion mutations into the E6 or E7 gene. (where E6 and E7 are the HPV oncogenes). This results in the induction of p53 or retinoblastoma protein (Rb), leading to cell cycle arrest and eventual cell death. Both HPV-16- and HPV-18-transformed cells were found to be responsive to targeted HPV genome-specific DNA cleavage (Kennedy et al, 2014).

4.       HIV/AIDS

Human immunodeficiency virus, commonly referred to as HIV, is a virus that attacks the body’s immune system. HIV infects CD4 T-lymphocytes, a type of immune cell that is important for fighting infections. HIV makes copies of itself inside the CD4 cell and then kills the cell, releasing more copies of the virus to infect and kill other CD4 cells. In experimental treatments, the approach is to use CRISPR genome-editing molecules to target the HIV DNA sequence stored in the host cell genome. The guide RNAs direct the Cas9 protein to cut at two sites within the HIV genome, surgically excising most of the genome and effectively eliminating HIV from the cell. The CRISPR treatment will be delivered by an AAV9 viral vector and administered as in vivo treatment by infusion.

5.       Blindness

Leber Congenital Amaurosis (LCA) is the most common cause of inherited childhood blindness, and LCA10 is the most common form of LCA. This disease is caused by a single nucleotide mutation in a photoreceptor gene, leading to serious vision loss or blindness within the first few months of life.

Many hereditary forms of blindness are caused by a specific genetic mutation, making it easy to use CRISPR-Cas9 to treat it by targeting and modifying a single gene. In addition, the activity of the immune system is limited in the eye, which can circumvent any problems related to the body rejecting the treatment. If enough cells are edited to make the healthy protein, the hope is that patients will regain vision.

6.       Huntington’s Disease

Huntington’s disease is a neurodegenerative condition with a strong genetic component. The disease is caused by an abnormal repetition of a certain DNA sequence within the huntingtin gene. The higher the number of copies, the earlier the disease will manifest itself.

Scientist have used this technology efficiently in preclinical studies, include targeting and inhibition of the mutant gene both in vivo and in patient-derived disease models. A research group at the University of Illinois, reported the application of this technology using Cas9 endonuclease from Staphylococcus aureus (SaCas9). The SaCas9 coupled with a single guide RNA disrupted the mutation in vivo, alleviated motor deficit symptoms, and even increased lifespan in R6/2 mice, a particularly aggressive murine model for Huntington's Disease. Results from this study are encouraging and point to the future use of CRISPR/Cas9 in treating Huntington's Disease in clinical settings.

Major Delivery System

The simplicity and accuracy of CRISPR-Cas9 made genome editing much more approachable for correcting genetic mutation or disrupting a target gene. The delivery of genome editing tools is one of the major challenges for in vivo genome editing. It can be classified into three major groups based on cellular entry mechanism: (i) biological methods, (ii) chemical methods, and (iii) physical methods.

The biological methods utilize natural biological materials, such as viral protein, peptide, or cellular receptor/membrane to mediate cellular entry. This category includes viral vectors, virus-like particles (VLPs), and cell-penetrating peptides (CPPs). The chemical methods use artificially synthesized materials, such as polymers, lipids, or metal, to catalyze cellular entry. These include liposomes, gold-nanoparticles (AuNPs), and lipid nanoparticles (LNPs). Physical methods rely on the physical energy of electricity or ultrasound to deliver the genes into cells. This category includes electroporation, sonoporation, and microinjection (Taha et al., 2022).

Figure 1: Various methods for delivering CRISPR-Cas9 components.

CRISPR/Cas9 Services

Nowadays, the technology of CRISPR/Cas9 has gained a massive interest of the scientists all around the world and has been applied many model diseases. Seattle Genova provides a broad range of products and services about CRISPR/Cas9 for our clients.

1) gRNA Design and Vector Construction Service

Careful design and construction of guide RNAs (gRNAs) and homologous recombination (HR) donors is an important first step in CRISPR/Cas9 technology. Seattle Genova can help you handle this first step to save yourself time and trouble. We offer target sequence cloning service into appropriate vectors, including all in one CRISPR/Cas, T7 vector, and lentiviral transfer vectors. There are two options for you:

You can provide the target sequences, choose a vector and we construct the plasmid.

For convenience, you can just provide genome sequence and we design the target sequences using our proprietary gRNA design tool.

We usually offer two gRNAs at least, and targets can destroy important domains of the protein and all the alternatively spliced transcripts. The knockout efficiency of our vector is very high.

2) CRISPR/Cas9 Cell Line Engineering

Seattle Genova provides a variety of reliable CRISPR/Cas9 cell lines engineering services to produce a genetically modified cell using any mammalian cell line and targeting any gene.

One-stop Service: From gene synthesis, design of gRNAs, cell transfection, to screening single clones, you can entrust all of these steps to us.

Wide Applications: Our services can apply to targets of any genes and mammalian cells.

Guaranteed Quality: We will validate the efficiency of full-allelic knock-out cell lines at mRNA or protein level upon request.

3) CRISPR/Cas9 Genome Knockout Kits

Seattle Genova offers genome-wide CRISPR gene knockout /knockin kits containing 2 gRNA vectors and donor DNA to help you modify the specific gene by yourself. The kits are ready-to-use and highly efficient with non-homology mediated gene knockout.

4) Transgenic Mice Services

Seattle Genova can also offer transgenic mice with CRISPR/Cas9-mediated genome modifications. Generation of knockout or knock-in mice using CRISPR/Cas9 in embryonic stem (ES) cell. (Burgio, 2018)

Figure 2: Generation of knockout or knock-in mice using CRISPR/Cas9 in embryonic stem (ES) cell. (Burgio, 2018)

Features of Our Services

·         Cost-effective

·         High efficiency

·         High specificity

·         Fast

·         Best after-sale service

CRISPR/Cas9 is a simple and efficient genome editing tool. Aided by our well-established platforms and experienced scientists, Seattle Genova has successfully completed dozens of genome engineering projects using CRISPR/Cas9. Because each project is different, if you don't see the CRISPR/Cas9 service you need above, please contact us, we can customize our offering to meet your specific project needs.

References

• Frangoul, H., Ho, T. W., & Corbacioglu, S. (2021). CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia. Reply. The New England journal of medicine, 384(23), e91. https://doi.org/10.1056/NEJMc2103481

• Kennedy, E. M., Kornepati, A. V., Goldstein, M., Bogerd, H. P., Poling, B. C., Whisnant, A. W., Kastan, M. B., & Cullen, B. R. (2014). Inactivation of the human papillomavirus E6 or E7 gene in cervical carcinoma cells by using a bacterial CRISPR/Cas RNA-guided endonuclease. Journal of virology, 88(20), 11965–11972. https://doi.org/10.1128/JVI.01879-14

• Taha, E. A., Lee, J., & Hotta, A. (2022). Delivery of CRISPR-Cas tools for in vivo genome editing therapy: Trends and challenges. Journal of controlled release: official journal of the Controlled Release Society, 342, 345–361. https://doi.org/10.1016/j.jconrel.2022.01.013