A cure for HIV is not impossible — Silencio Bio project proposal

Curing HIV with gene editing technologies.

The Problem of HIV

In 2019, 38 million people globally were living with HIV. Out of that number, 690 000 people have died from AIDS-related illnesses. In that same year, 1.7 million people became newly infected with HIV [1]. Although Sub-Saharan Africa is the hardest-hit region, where the people infected account for 68% of the global population of HIV [2], approximately 1.2 million people are still living in the United States with HIV, with an estimated 36,400 new cases in 2018. This means that the problem of HIV is still prevalent in North America [3].

Although when people think of HIV, they immediately think of the 80s and the 90s, it’s important to note that we are still in the 30 year-long HIV epidemic. We have been in the COVID-19 pandemic for a little over a year and we already have a vaccine. But even 30 years in, we still don’t have a vaccine or a cure for HIV. Thus, given the global statistics, the HIV epidemic is still a prevalent problem to human society and a daunting challenge for the medical community.

Antiretroviral Therapy

The only current HIV treatment, antiretroviral therapy (ART), was accessed by 26 million people at the end of June 2020. This treatment prevents the virus from multiplying which reduces the amount of HIV in the body and reduces transmission. Between 2010 and 2019, HIV infections have declined by 23% and since the peak in 1998, infections have been reduced by 40% [1]. However, ART is not a cure and does not effectively free the body completely from the long-term consequence of HIV. The cost of ART is between $1,080 to $2,700 per month for the rest of a patient’s life which is expensive [4].

ART is a medication regimen composed of different combinations of 17 drugs to slow the progress of the virus and reduce transmission. A combination of 3 or more types of drugs is used to target the virus at different stages of its life cycle. The regimen starts immediately after diagnosis and is tailored to the individual requirements of the patients such as their medical history [4].

The classification of ART drugs are usually done using the classes of Nucleoside reverse transcriptase inhibitors (NRTIs), Non-nucleoside reverse transcriptase inhibitors (NNRTIs), Protease inhibitors (PIs), Integrase inhibitors (INSTIs), Fusion inhibitors (FIs), Chemokine receptor antagonists (CCR5 antagonists), Post-attachment entry inhibitors, Pharmacokinetic enhancers, Complete regimen combination ARTs. Each type of drug targets the virus at a different stage in its life cycle [4].

Nevertheless, ART is no cure for HIV and it has some serious limitations.

1. ART is a life-long therapy that can keep HIV patients healthy for many years. Medication use for ART should never be stopped even if the virus load becomes undetectable, otherwise, the virus is likely to mutate and become drug-resistant [4].
2. Although the widely used drugs for ART are well tolerated, most of them have some short-term toxic effects and all have the potential for both known and unknown long-term toxic effects [5].
3. Minimal or full control of viral replication does not restore health, ART will just slow down the progression of HIV, but your immune system still becomes weaker. Studies show that patients who have been following an effective long-term regimen often show persistent immune dysfunction and they have a higher risk for various non-AIDS-related complications, including heart, bone, liver, kidney, and neurocognitive diseases [5].

CRISPRoff

A short summary and illustration on how CRISPRoff works and some results from significant experiments.

Recently, a study by Nuñez et al., 2021 was published discussing a new CRISPR technology called CRISPR-off. Instead of the current CRISPR/CAS-9 process which cuts strands of DNA to make double-stranded breaks and leaves permanent changes to the cell genome, CRISPR-off gives us the ability to silence a variety of genes and essentially stop them from affecting the body.

The technology consists of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modification, allowing researchers to control gene expression. A single-guide RNA (sgRNA) guides CRISPRoff to its target gene. CRISPRon can reverse the changes of CRISPRoff by demethylation; compared to CRISPR/Cas9, CRISPRoff is a safer tool for making epigenetic changes because it does not damage DNA and it is reversible. An easy way to explain the difference between CRISPRoff and CRISPR/cas9 is that if the lights in your room are too bright for you and you want to turn them off, instead of taking a baseball bat and shattering all the light bulbs with little precision, you use a switch to turn your light on and off when you want to.

CRISPRoff can silence most genes, including those without CpG islands and those that control gene expression but do not code for protein.

The epigenetic changes made by CRISPRoff were found to be heritable and that the epigenetic changes persist through cell division and differentiation. When CD81 was silenced by CRISPRoff in induced pluripotent stem cells (iPSCs) that then went through neuronal differentiation, 90% of the differentiated iPSCs had a silenced CD81. Thus, the transient expression of CRISPRoff writes specific epigenetic changes that are memorized by cells through cell division and differentiation. An easy way to explain the difference between CRISPRoff and CRISPR/cas9 is that if the lights in your room are too bright for you and you want to turn them off, instead of taking a baseball bat and shattering all the light bulbs with little precision, you use a switch to turn your light on and off when you want to.

Furthermore, after 10 days of differentiation of iPSC-derived neurons with a silenced MAPT, a gene encoding the Tau protein involved in various neurological diseases, or a non-targeting control, it was found that there were ~ 30% of cells with reduced Tau expression compared to a non-targeting control. This result signifies that epigenome editing with CRISPRoff to modulate gene expression in cells where delivery of gene editing platforms remains a challenge is possible.

The authors have identified some limitations of this study that need to be addressed with more research:

1. Most of the CRISPRoff experiments and the genome-wide screens were done using sgRNAs predicted to be optimal for CRISPRi. Future efforts to design more optimal sgRNAs will increase the efficiency of gene silencing activity.
2. The experiments were done in polyclonal cell lines. An exploration of potential clonal differences in methylation and its effects on gene silencing and potential off-target sites should be done.
3. It remains unknown what genes are amenable to stable or metastable silencing. More research needs to be done to identify the regulatory elements that can make programmed epigenetic changes more stable in all genes [6].

HIVoff: CRISPRoff to Silence HIV-1 DNA

We propose a cure to HIV-1 by using CRISPR-off to silence the integrated proviral HIV-1 DNA. We hypothesize that although the HIV-1 DNA remains in the host cells, the silencing of the DNA would stop the virus from affecting the host. Essentially, this should cure the infected individual of HIV. We base our hypothesis on a similar study by Dash et al., 2019 that instead used AAV9-delivered CRISPR/Cas9 to cut out the proviral HIV DNA from the helper T cells (CD4) in the lymph node, liver, lung, brain, spleen & gut of humanized mice. This experiment was done alongside the administration of LASER ART which is a type of art that employs long-acting hydrophobic lipophilic antiretroviral nanoparticles that have a higher inhibitory effect than standard ART. This study split the mice into three cohorts where one cohort only received LASER ART, the second received only CRISPRoff and the third received both LASER ART and CRISPRoff. The researchers cured 30% of all the humanized mice from HIV-1. To reduce the chance of dangerous mutations forming as a result of random DNA repair, we propose a similar cure but with CRISPRoff. Cutting DNA with CRISPR/Cas9 in humans has a lot more risks attached to it. This is why our proposal requires the use of CRISPRoff instead to make the therapy much safer for humans. Going back to our light bulb analogy, if you use a switch to turn the lights on and off, it is much safer than breaking the light bulbs because you won’t have glass shards all over your room afterwards.

A summary of the Dash et al., 2019 study and a short comparison with our study.

To test the feasibility of our proposed cure, we are outlining a preclinical research proposal based on the methodology used in the Dash et al., 2019 study [7].

Although the study by Dash et al., 2019 provides our proposal with a plausible methodology, there are still a number of ambiguities in our proposal for a cure that need to be addressed by a preclinical study, given that CRISPRoff is such a new technology with different mechanisms than CRISPR/Cas9. These ambiguities are:

1. Will the silencing of the proviral DNA cure the infected individual of the effects of HIV-1 (viremia) on their body and stop the progression towards AIDS?
2. Does LASER ART have to be continued after CRISPRoff treatment?
3. How many doses of the CRISPRoff therapy need to be administered until all the infected helper T cells are treated? What should be the time interval between these doses to ensure durable memory of gene silencing?

Outline of Preclinical Study

Our preclinical study consists of 3 rounds. To see if LASER ART should be administered during and after CRISPRoff treatment, 3 cohorts of mice are used in rounds 1 and 2(just LASER ART, just CRISPRoff, LASER ART and CRISPRoff).

1/ We want to use sample groups of humanized mice (3 rounds of the experiment) → starting with round 1 of mice:

• Humanized helper T-cells (CD4) are infected with HIV outside of the mice in the lab and then injected into the 3 cohorts of mice (cohort 1: receives just LASER ART, cohort 2: receives just CRISPRoff, cohort 3: receives both LASER ART and CRISPRoff → the outcome is to monitor if LASER ART needs to be administered during CRISPRoff treatment and after).
• CRISPRoff is guided to the CD4 cells with a sgRNA targeting the LTR1 and GagD. The viral vector used is AAV9 because it has robust transduction efficiencies in multiple tissues including the central nervous system which is a significant putative reservoir for HIV-1. AAV9 is delivered in vivo to infected organs (lymph node, liver, lung, brain, spleen & gut).
• Test round 1 of mice and to see if the HIV DNA is being expressed. To do the testing, we will use ultrasensitive semi-nested real-time qPCR with primers and probes designed for detection of HIV-1 gag and then confirm by digital droplet PCR (ddPCR).

2/ The cured CD4 cells from round 1 of mice are then transferred to round 2 of 3 cohorts of non-infected mice to see if the new mice will express the HIV-1 DNA or not.

3/ With round 3 of mice, the experiment from round 1 of mice is repeated to see if the CRISPRoff will once again fully silence virus DNA and to determine how many doses of CRISPRoff need to be administered and with what time interval should they be administered until no HIV-1 DNA is expressed and there is durable memory of gene silencing in round 3 of mice.

You can find the exact methodology we want to employ in this link [7].

Comparison of ART VS CRISPRoff silencing

ART is not a cure for HIV, it is a treatment. Given that ART does not eliminate the problem of HIV from the roots, we cannot compare its benefits with our proposal which uses CRISPRoff to silence the gene expression of the proviral HIV-1 DNA that will stop the effects of the virus on the infected individual. It is important to note that although CRISPRoff does not remove HIV-1 from the patient, it stops the replication and activities of the virus within the body. Thus, the infected patient is alleviated from any symptoms and risks associated with HIV.

The experiment by Dash et al., 2019 cured 30% of the mice by using CRISPR/Cas9 [7]. We predict that this number can be higher if CRISPRoff is used because an experiment by Nuñez et al., 2021 silenced Snrpn-GFP in 80% of transfected cells and HIST2H2BE in 90% of transfected cells after at least 50 days [6]. Therefore, a 30% success rate is the baseline for our preclinical study.

Clinical Trials & FDA Approval

Image from the FDA website.

There are various phases of the clinical trials prior to final FDA Approval.

• Phase 0 of a clinical trial is done with a very small number of people, usually fewer than 15. Investigators use a very small dose of medication to make sure it isn’t harmful to humans before they start using it in higher doses for later phases. If the medication acts differently than expected, the investigators will likely do some additional preclinical research before deciding whether to continue the trial.
• During phase I of a clinical trial, investigators spend several months looking at the effects of the medication on about 20 to 80 people who have no underlying health conditions. This phase aims to figure out the highest dose humans can take without serious side effects. Investigators monitor participants very closely to see how their bodies react to the medication during this phase. According to the FDA, approximately 70 percent of medications move on to phase II.
• Phase II of a clinical trial involves several hundred participants who are living with the condition that the new medication is meant to treat. They’re usually given the same dose that was found to be safe in the previous phase. Investigators monitor participants for several months or years to see how effective the medication is and to gather more information about any side effects it might cause. The FDA estimates that about 33 percent of medications move on to phase III.
• Phase III of a clinical trial usually involves up to 3,000 participants who have the condition that the new medication is meant to treat. Trials in this phase can last for several years. The purpose of phase III is to evaluate how the new medication works in comparison to existing medications for the same condition. To move forward with the trial, investigators need to demonstrate that the medication is at least as safe and effective as existing treatment options. To do this, investigators use a process called randomization. This involves randomly choosing some participants to receive the new medication and others to receive an existing medication. Roughly 25 to 30 percent of medications move on to phase IV.
• Phase IV clinical trials happen after the FDA has approved medication. This phase involves thousands of participants and can last for many years. Investigators use this phase to get more information about the medication’s long-term safety, effectiveness, and any other benefits [8].

Cost Breakdown

Clinical Trials Cost

The average cost of phase 1, 2, and 3 clinical trials across therapeutic areas is around $4, 13, and 20 million respectively. The total cost of development for HIVoff will be $37 million. Pivotal (phase 3) studies for new drugs approved by the Food and Drug Administration (FDA) of the United States cost a median of $41,117 per patient.

Treatment Cost

Based on research from current gene therapy costs, the average cost for our HIV-off treatment would be close to 2 million dollars. Although expensive, this solution is an extremely effective cure. Over time we will be able to lower costs, but as a starting point we must use it accurately, therefore leaving us with this cost

Cost Comparison

Antiretroviral therapy cost

HIV care involves a type of medication called antiretroviral therapy (ART) and regular visits with your doctor. One study estimated that costs of this care could run anywhere between $1,800 to $4,500 each month during a person’s lifetime. Most of this, about 60%, comes from the high cost of ART medications [9].

Current gene therapy costs

In 2019, Novartis announced the launch of Zolgensma, a one-time virally-delivered gene therapy designed to provide a fully functional copy of the faulty SMN1 gene that causes the disease. But here’s the catch: Novartis has priced the gene therapy at $2 million per treatment. Given the prior successes of CRISPR and Spark Therapeutics in treating hereditary retinal and blood disorders with gene therapy, Editas’s candidates seem very promising. Indeed, if it can advance them to approval, it will have huge market opportunities ahead. For example, Spark Therapeutics’ Luxturna costs $425,000 per eye treated. Meanwhile, novel drugs for SCD sell for between $7,000 to $15,000 per course of therapy [10].

Outcome

To start, we would have to start the rollout in richer countries because they have the infrastructures, we calculate that baseline we would reach 360 000 people in the US alone. At least globally, these many people will be able to afford such a therapy in the early stages. This will bring down the urgency of the HIV epidemic and once rich countries have the burden of HIV treatment costs alleviate from their economy, those fundings for ART accessibility in rich countries can go towards poor countries. Consequently, an indirect impact of a cure in rich countries would be to move current HIV treatments to poor countries that have more cases and fewer resources.

Considering that there is no commercial gene editing therapy yet approved for commercial use, we predict that the time for development and FDA approval can exceed 10+ years. However, the long-term benefits of such a technology are worth the long wait. We hypothesize that if another retroviral epidemic were to take place again in the next century, we would have the technology for a cure prepared if we work on one for HIV right now. Therefore, our solution goes beyond the problem of the 30 years long HIV epidemic and introduces hope for future epidemics.

This article was written by Diba D. (The Problem of HIV → Comparison of ART VS CRISPRoff silencing + Outcome) and Siyana L. (Clinical Trials and FDA Approval → Cost Comparison).

References

1. UNAIDS. (2020). Global HIV & AIDS statistics — 2020 fact sheet. https://www.unaids.org/en/sorrypagenotfound
2. Global HIV and AIDS statistics. (2020, February 18). Avert. https://www.avert.org/global-hiv-and-aids-statistics
3. U.S. Statistics. (2021, April 21). HIV.Gov. https://www.hiv.gov/hiv-basics/overview/data-and-trends/statistics#:~:text=The estimated number of HIV,among all other age groups.
4. How Effective is antiretroviral Therapy (ART) for HIV? (2020, July 6). MedicineNet. https://www.medicinenet.com/how_effective_is_art_for_hiv_infection/article.htm
5. Volberding, P. A., & Deeks, S. G. (2010). Antiretroviral therapy and management of HIV infection. Lancet (London, England), 376(9734), 49–62. https://doi.org/10.1016/S0140-6736(10)60676-9
6. Nuñez, J. K., Chen, J., Pommier, G. C., Cogan, J. Z., Replogle, J. M., Adriaens, C., Ramadoss, G. N., Shi, Q., Hung, K. L., Samelson, A. J., Pogson, A. N., Kim, J., Chung, A., Leonetti, M. D., Chang, H. Y., Kampmann, M., Bernstein, B. E., Hovestadt, V., Gilbert, L. A., & Weissman, J. S. (2021). Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing. Cell, S0092–8674(21)00353–6. Advance online publication. https://doi.org/10.1016/j.cell.2021.03.025
7. Dash, P.K., Kaminski, R., Bella, R. et al. Sequential LASER ART and CRISPR Treatments Eliminate HIV-1 in a Subset of Infected Humanized Mice. Nat Commun 10, 2753 (2019). https://doi.org/10.1038/s41467-019-10366-y
8. What Are Clinical Trials and Studies? (2020). National Institute on Aging. https://www.nia.nih.gov/health/what-are-clinical-trials-and-studies
9. Rachel, R. N., & Carter, A. C. (2019). The Cost of HIV Treatment. Healthline.Com. https://www.healthline.com/health/hiv-aids/cost-of-treatment
10. Wong, C. H. (2020, January 1). Estimating the Financial Impact of Gene Therapy*. MedRxiv. https://www.medrxiv.org/content/10.1101/2020.10.27.20220871v1.full (this is a preprint paper)