CarcinoGene: Preventing Breast Cancer with Gene Therapy for BRCA1 Mutations
It is estimated that one in eight women will be diagnosed with breast cancer in her lifetime. That’s a crazy number. Now, on top of this, women with genetic mutations have a higher lifetime risk of the disease. It’s estimated that 55–65% of women with a mutation in BRCA1 will develop breast cancer before age 70.
Mutated BRCA genes may increase people’s risk for cancers such as ovarian, prostate, and pancreatic cancer. There is also an increased likelihood of developing cancer at an early age, developing breast cancer in both breasts, or developing more than one type of cancer in their lifetime.
In this article, I’ll be discussing using gene therapy for Breast Cancer gene 1 mutations because the need for a gene therapy solution for BRCA1 mutations, which leads to triple-negative breast cancer, is more significant than Breast Cancer gene 2 given that current cancer therapies are less efficient in BRCA1 mutations. This is because BRCA1 mutations are estrogen receptor-negative, progesterone receptor-negative, and HER2 new negative.
Heredity
Everyone has two copies of this gene, one copy being inherited from each parent. Inheriting harmful variants leads to mutations in this gene and therefore results in an increased risk of cancers, most significantly for breast and ovarian cancer. The harmful variant can be inherited from either parent, and these mutations are known as germline variants meaning the variation is present in all the cells of the body from birth.
BRCA1 Gene Function
The BRCA1 gene is located on chromosome 17 in the hg38 human genome. The BRCA1 gene encodes a nuclear phosphoprotein. This protein a) maintains genomic stability and b) acts as tumour suppressors, c) produces a protein that repairs DNA.
The protein combines with tumour suppressors, DNA damage sensors, and signal transducers to form a large protein complex from the BRCA1-associated genome surveillance complex. This protein plays an active role in transcription, DNA repair of double-stranded breaks, and recombination because the gene product associates with DNA polymerase II and interacts with histone deacetylase complexes through the C-terminal domain.
BRC1 and Cancer
The craziest part is that mutations in the BRCA1 gene are responsible for around 40% of inherited breast cancers & over 80% of inherited breast and ovarian cancer.
This chart shows the increased risk of malignancies in different types of cancer when there is a variation in the BRCA1 gene.
Current Treatments
So what’s being done today?
Here are 3 main treatment options available to patients in hospitals:
- Radiation therapy: using high doses of radiation to kill cancer cells. with radiation therapy, there’s an inability to kill tumour cells that cannot be seen on imaging scans and are therefore not always included on the 3D models.
- Surgery: an operation where doctors cut out cancer tissue. In surgery, the whole tumour is not able to be removed sometimes.
- Chemotherapy: Using special medicines to shrink or kill the cancer cells. With chemotherapy, there is often the damage caused to surrounding tissues (e.g. lung, heart), depending on how close the area of interest is located to the tumour.
Our Solution
The solution we propose at Carcinogene is a preventative gene therapy for the BRCA1 gene.
Our solution consists of 3 steps:
- DNA Sequencing of the patient.
- Creation of personalized GRNA and plasmid & DNA template.
- Delivery of CRISPR Cas9 to the Patient & cancer cells
In order to explain the methodology we want to employ at CancinoGene to edit mutated BRCA1 and BRCA2 genes, we have demonstrated our protocols on a simulation carried out on Benchling.
1. DNA Sequencing of the patient
To detect what mutation the patient has, we use New Generation Sequencing (NGS).
There are three parts to this process
- Isolation
- Amplification
- Sequencing
We Isolate the BRCA1 gene and duplicate it many times so we have efficient copies and then sequence it so we have the base letter; what we’re doing is taking analyzed bases from the person and comparing it to known/studied mutations of the BRCA1 gene. By comparing these 2 DNA sequences we can see where the mutation actually is. Tumour-specific DNA rearrangements could be detected in the patient’s plasma, suggesting that NGS could be used to personalize the monitoring of the disease.
In our simulation, we uploaded the DNA sequence of the mutated BRCA1 gene from the NCBI database.
2. Creation of personalized GRNA and plasmid & DNA template
Once we identify the mutation in the genome of the patient, we begin to design a gRNA for the target sequence. In this example, we are looking at a patient with a pathogenic variant who has a 1,508 bp deletion on a part of exon 23 of BRCA1. This deletion partially removes the C-terminal BRCT domain of the BRCA1 protein, which is important for DNA repair activity. So while this deletion does not create nonsense-mediated decay, it will change the BRCA1 protein function.
To design the gRNA, we can use Benchling to find optimal target sites. We select exon 23 and evaluate the possible sgRNAs. We are using a single guide of wild-type Cas9 with a Pam sequence of NGG from SpCas9, 3’ side. We use a Cas9 enzyme because we are editing a DNA sequence. Optimally, we aim for an off-target score of 50 and an on-target score of 60, however, the best gRNA for this region has an on-target score of 24.9 and an off-target score of 44.3. At CancinoGene, our goal is to utilize the most efficient gRNA designs to reduce the chance of off-target edits.
Once we have designed the personalized gRNA, we will transfect a lentiCRISPRv2 plasmid with the gRNA/cas9 complex. This is the preview of the BRCA1 plasmid we have created to edit the deletion on exon 23. The highlighted region on the right shows the placement of the designed gRNA within the plasmid.
In our methodology, we use homologous repair (HR) to repair the double-stranded break. To make the HR template for the 1,508 bp deletion on a part of exon 23, we design an HR arm of range 77789 to 79297. In this particular HR template, we have included the guide sequence of the gRNA because the deletion begins in the middle of the guide sequence. What this means is that we will be re-inserting the correct base pairs before the deletion begins. The following HR template is now ready for de novo synthesis.
3. Delivery of CRISPR Cas9 to the Patient & cancer cells
Once the Cas9/sgRNA and DNA template have been synthesized through De Novo synthesis, they will be delivered to the patient in vivo through lentiviral transduction. The first lentivirus containing the lentiCRISPRv2 plasmid is cultured, then they are injected into Mammary cells in multiple doses. After treatment, NGS of a blood sample is used to measure the efficiency of the lentivirus injections and measure the change in mutations. The patient will be monitored for immune reactions towards the lentivirus. The treatment will have to be repeated in a year.
Why Gene Therapy?
Without a gene therapy solution to prevent tumour growth in women with BRCA1 mutations in the first place, these women will be recommended to receive bilateral mastectomies if they have just been diagnosed with breast cancer.
Breast cancer is pretty common, so many women will undergo a single or double mastectomy in their lifetime. Having a mastectomy will give you the best chance of a positive outcome. However, preparing for your procedure and getting used to your body after a mastectomy can take a significant toll on you.
Studies show evidence that mastectomies create an emotional toll on women and often result in the occurrence of post-surgery depression, and self-esteem issues.
Those who test positive for a mutation in the BRCA1 gene will often hand down that variant from generation to generation. Handing down the troubles of breast cancer. But that can all stop right here.
The Impact
Every 2 minutes a woman gets diagnosed with breast cancer worldwide, and in the United States, every 13 minutes a woman dies of breast cancer.
Our solution is a step forward. It’s a step forward to eliminate the suffering and pain caused by cancer, an ugly word with a really scary meaning.
Our team supports those who are struggling with breast cancer, admires those who have survived the illness while honouring those who have been taken from us. But we will never, ever give up our fight to come up with a cure.
