Final Presentation

Script

Matt

Good evening. My name is Matt Gethers, my group members are Yi Wang, Rob Warden, Stephanie Nix, Courtney Lane, Jessie Wang, and David Ying.

We are here today to present to you a novel treatment for HIV infection. This treatment involves injecting HIV patients with engineered hematopoietic stem cells that will become a self-replicating pool of erythrocytes, or red blood cells. These manipulated red blood cells act as viral sinks to deplete the viral load.

[Slide 2 – Clinical Background]

HIV kills 2.1 million people a year.
In Boston and the rest of the U.S., about one in a hundred people are infected, whereas in some sub-Saharan African countries, over 30% of the population is infected.
Current techniques for treating HIV, such as nucleoside analogues and reverse transcriptase inhibitors, decrease the viral load of HIV to less than 50 virions/mL, delaying the onset of AIDS for potentially many years.
However, treatments are expensive and require frequent doses.
A new therapy for HIV is necessary which costs less and requires fewer treatments – ideally, only one.

[Slide 3 - RBC Viral Trap]

Our solution is an erythrocyte-based viral trap called HIVac.
As opposed to all current small-molecule based HIV drugs, we want something that is renewable to avoid the need for frequent dosing.
Hence we have chosen a cell-based mechanism, because certain cells can replicate themselves.
Because we decided to go with a cell-based approach, we had a lot of options at our disposal.
We decided to exploit HIV's mechanism of infection by providing a decoy.
An ideal decoy is a cell that would not support any other part of the viral life cycle after infection.
Red blood cells are ideal for this application because they lack a genome and thus cannot support viral replication. To mediate viral infection, you need only express CD4 and chemokine co-receptors on the cell surface. They are also abundant in the body and greatly outnumber T-cells, the primary tropism of HIV.
The engineered cells could then be introduced to the patient.

[Slide 4 - Engineering Hematopoeitic Progenitors]

But remember, our main design goal is to treat HIV with ideally one dose and the red blood cell lifetime is finite. Also they have no genome and so they don't replicate themselves.
To achieve a permanent effect, we will engineer the red blood cell progenitors and place them in the patient. These cells would renew themselves and differentiate into the viral sinks.

[Slide 5 - Therapy Diagram]

Obtain Patient Stem Cells: The first step of the therapy is to collect hematopoietic stem cells from the patient. By taking cells from the patient, we can avoid immune rejection.
Reengineering: The second step is to engineer the stem cells to express viral receptors upon differentiation into red blood cells.
Introduce engineered stem cells to patient: We will then reintroduce the cells into the patient.
Erythropoiesis: After replacement in the blood stream, the stem cells will differentiate into red blood cell viral sinks.
HIV infects decoy RBCs: HIV infects the red blood cell viral sink and is unable to replicate.
Exponential titer reduction: Given the abundance of red blood cells in the body, it is more likely for a virion to encounter a viral sink than a T-cell.
Viral control: This will lead to control of viremia.

Rob: Design Detail

Slide 6:

In order to create our HIVac cells, we will need to significantly alter the DNA of the patient's hematapoietic stem cells. We will introduce these genes by infecting stem cells with lentiviruses carrying the appropriate genes.

Slide 7:

Our first design goal was to make our HIVac cells infectable. We do this by adding CD4, CCR5, and CXCR4 under an erythrocyte specific promoter, ankryn-1. These proteins are all that is necessary for viral fusion, and therefore all that is necessary to produce our sink. However, the changes can't stop there.

Slide 8:

HIVac is a completely new class of therapeutic, so ensuring patient safety is another fundamental aspect of our design. An RNAse is the first of several safety features that we will include in HIVac cells. By also expressing the RNAse under ankryn-1, we will completely destroy the HIV genome after infection, so that there is no chance of escape. As described in the design paper, the RNAse will be engineered to include a variety of other regulatory features as well, so that it minimally disrupts other functions in the cell.

Slide 9:

Our next safety feature will protect against HIV takeover while the cell is producing receptors but has not yet shed its nucleus. The literature shows that the use of 4 short hairpin RNAs, each targeting a different highly conserved region of the HIV genome, can silence HIV transcription even with its high mutation rate.

Slide 10:

Next, we will add a suicide gene, as a last-resort fail-safe mechanism. Herpes Symplex Virus thymadine kinase, HSV-tk for short, is a commonly used suicide gene that makes a cell supersensitive to the drug gancyclovir. With this, we can ablate the stem cell population should the patient show signs of treatment complications, such as graft versus host disease.

Slide 11:

Finally, we will force the stem cells to undergo erythropoiesis. This accomplishes two goals. First, it limits the scope and reach of our novel system. Second, it should increase the rate of erythropoiesis so that fewer injected stem cells are necessary. This can be done by knocking down two genes with shRNA, PU.1 and Fli-1, transcription factors that control cell fate decisions. With these genetic changes, our HIVac cells should be capable of safely trapping and destroying blood-borne HIV.

Slide 12:

Our final challenge during HIVac design was to show it could work. So we created a model which accounted for changes in HIVac, HIV, and T-Cell populations. As we show here, even at the height of infection, HIV counts drop dramatically shortly after the treatment is administered and control the virus at least as well as current treatments after four months. And this is all after just ONE treatment. The model also shows us that our HIVac population levels out at about 1% of natural erythrocytes, as well as a steady increase in healthy CD4+ T-Cells.

Timeline for Development

Our design for an erythrocyte viral trap is unique - there is nothing like it on the market right now, or even anything in development.
However, there does happen to be a patent on the idea of an erythrocyte viral sink issued less than a year ago.
In order to secure the intellectual property for HIVac, we will need to acquire this patent at the very beginning.
We will then move onto engineering our product with the features we discussed previously, testing it, and then getting FDA approval.

Once approved, we plan on initially marketing only in the first world.
We will set a high initial price close to $100,000 to pay for our development costs.
While this price seems high, it should be compared to the $25,000 annual cost of HAART treatment.
HIVac is a one-time, permanent treatment, so it could potentially save money for patients while also being more effective.
A conservative but crude estimate puts the number of HIV patients who could afford this price at approximately 100,000 in the U.S., and double that including Western Europe and Canada.
For every patient we treat we will store a sample of their engineered HIVac cells, establishing a library of stem cells with different HLA alleles.
This will lower the cost for future patients who can find an HLA-match to a stored stem cell in our library.

After demonstrating the efficacy of HIVac in the first world, we will expand HIVac to Sub-Saharan Africa and the rest of the third world.
We will seek grants from philanthropic organizations or governments to treat an initial group of HIV patients in a given country or area.
Using our previous stem cell library idea, we will establish a more genetically relevant stem cell database in each country or region with local HLA alleles.
This will allow us to lower treatment costs to the price range of annual HAART treatment in the third-world, around $500.

Matt Gethers/20.380 HIV Project/Final Presentation

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