Matt Gethers/20.380 HIV Project/Design Pitch

=Design Pitch=

Design (Rob and Matt)

 * One problem with HIV treatment is the need to take expensive drugs all your life. Even if we couldn't eliminate the virus from the body completely, controlling viremia at the current benchmark but with less effort on the part of the patient and provider would be a huge improvement.
 * Plan is to introduce a permanent viral sink to patients.
 * Rather than trying to inhibit a part of the viral lifecycle through a drug, allow HIV to infect cells, but we choose the target.
 * Functionalize the membranes of RBCs with CD4 and cytokine co-recpetors to mediate infection by HIV.
 * Upon fusion with the RBC, there is no genome in which to integrate or transcriptional/translational machinery.
 * RBC acts as "dead end" from which virus cannot emerge.
 * We want this treatment to remain in the body permanently, so we will collect hematopoetic progenitors from the blood stream, transfect CD4 and chemokine receptors under the control of promoters active during differentiation using a lentiviral vector. These cells will then be re-introduced to the patient where they will expand and act as a permanent source of viral sinks.

Benchmarks (Yi)

 * How low would the viral titer have to be in order to match current treatments (AZT)?

Modeling (Steph)

 * What processes might we want to model to determine if this project is worth taking forward?
 * Will want to test Yi's benchmark for efficacy (can we get viral titer this low).
 * Differentiation rate, Death/clearance rate, probability of infection of RBC as opposed to T-cell,selection.

In Vitro (Courtney-face)

 * In vitro tests for efficacy?

In Vivo (Jessie)

 * Animal models?

IP (David)

 * Previous work:
 * Europoean Patent No. EP0298280: Animal derived cell with antigenic protein introduced therein (1994), assignee is "HAPGOOD, C.V., a Netherlands Antilles Limited Partnership"
 * http://www.freepatentsonline.com/EP0298280.html
 * Basically same as US patent below
 * US Patent No. 5677176: Animal derived cell with antigenic protein introduced therein (1997)
 * http://www.google.com/patents?id=PAwpAAAAEBAJ&output=html
 * US version of Sheffield Medical Technologies from article (same title as claimed European patent) although Sheffield isn't mentioned anywhere
 * Claims an engineered RBC with CD4 on its surface, however claimed use is to encourage fusion of engineered RBC with HIV infected cells via syncytium formation
 * Engineered RBCs or liposomes contain a cytotoxic agent
 * Mechanism: infected cells will have gp120 on surface, which binds CD4 and allows for cell fusion with anything CD4+, which then kills both cells either via its cytotoxic load or splenic filtering of RBCs
 * Important: patent does not discuss any form of genetic engineering - they plan to incorporate CD4 into target RBCs basically by manually mixing concentrated CD4 and RBCs
 * Patent briefly mentions our "viral sink" idea (page 7, under "Clinical Use"), but this isn't in the claims and the authors say the syncytia formation is "more important"
 * US Patent No. 7462485: Modified erythrocytes and uses thereof (December 2008)
 * http://www.freepatentsonline.com/7462485.html
 * Inventor is Lawrence F. Glaser, who may be associated with ViraLogic Technology (as of 2002) but no record of this company exists anywhere
 * This patent basically covers our idea to the tee, almost
 * Focuses on a final treatment involving injection of RBCs, not progenitors
 * Mentions using hematopoietic cells, but unclear whether that would cover our application
 * Claim of interest: "20. A human hematopoietic progenitor cell comprising a recombinantly-produced sequence encoding CD4 or an HIV coreceptor." (Note: this claim was only on the application, and disappeared from the approved version)


 * How would we go about securing a patent/starting a company?
 * License the Glaser patent if we think it's needed?

Questions/Obstacles

 * What happens when you overexpress CD4+ in the body? Have any effects?

Yi
Good morning. My name is Yi Wang, my group members are Matt Gethers, Rob Warden, Stephanie Nix, Courtney Lane, Jessie Wang, and David Ying.
 * [Slide 1 – Intro]

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 – Epidemiology]


 * HIV kills 2.1 mill. people a year.
 * 1/100 people in the city of Boston live with HIV/AIDs.
 * In the United States, approximately 1% of the population is infected with HIV, whereas in some sub-Saharan African countries, over 30% of the population is infected.


 * [Slide 3 – Current Shortcomings]


 * 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.

Matt
[Slide 4 - RBC Viral Trap]


 * To get around the need for dosing, one approach is to develop a genetically-encoded therapeutic. That way, it would be capable of renewing itself indefinitely.
 * If we were to encode a mechanism that protects or cures cells of HIV infection, you're really talking about permanently modifying a large number of human cells. Given the problems associated with human genetic engineering, this approach isn't likely be widely accepted.
 * If you don't engineer cells to protect them from infection, another approach is to engineer a population of cells that are susceptible to viral infection, but incapable of supporting viral replication. These cells would then act as viral "sinks." If you put enough of these in the body, than it's plausible that you could control viremia.
 * 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 achieve similar viral titers with ideally one dose and the red blood cell lifetime is finite. After 120 days, they are removed in the spleen. Also remember that they have no genome and so don't replicate themselves.
 * To achieve a permanent effect, you could engineer the red blood cell progenitors and place them in the patient. These cells would renew themselves and differentiate into the viral sinks.

Add this: [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: Because these stem cells remain in the blood stream, we need only replace the cells in the blood.
 * 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
[Slide - Genomic Changes]

In order to do all this, we will have to make a few simple changes to the stem cell genome.

First, we will add the three receptors required for HIV infection under the GATA-1 promoter. GATA-1 is an erythrocyte specific promoter that is used to drive the production of hemoglobin.

Second, we plan to add RNAses under the GATA-1 promoter as well. These will ensure that the HIV genome is not only unusable, but completely destroyed. This way, if the cell is ruptured there is no chance of the HIV genome escaping the trap.

Next, we will add Anti-HIV siRNA. This is to prevent viral replication if the cells are infected before they shed their nuclei. It has been shown that targeting 4 HIV genes with siRNA will silence HIV translation even in the presence of mutations.

Also, we will give the cells a drug supersensitivity. This is a fail-safe mechanism that will be utilized to destroy the engineered population should something unexpected occurs.

Lastly, we will eliminate all other possible cell fates for the stem cells. This way, we can control and limit the engineered population. It may also increase the rate of erythropoiesis and therefore the efficacy of the treatment.

[Slide - Back-of-the-envelope Efficacy] (Change to Promising Initial Calculations)

While developing this idea, we did some initial calculations to check the trap's feasibility. Roughly speaking, each cubic millimeter of human blood contains 5 million red blood cells and 1,000 CD4+ T-Cells. Assuming that we can achieve an engineered population of 1% of the total red blood cell count, we will have a 50X excess of traps to T-Cells. Assuming HIV has no preference for either cell type, this means that viral production will be reduced by about 50x during each characteristic time period, the length of which has yet to be determined. Within three time periods, viral production should drop below an average of 1 viron/day.

Although our initial calculations support strong viral control at the very least, we will need to develop a better model to further support our idea. So now Stephanie will talk about the details of that model.

-make upregulation/down regulation more interesting (maybe a diagram of a cell?)

Steph
''note: if we have room on the slides, can we have a diagram instead? something like birth -> birth rate factors -> thing being modeled -> death rate factors -> death''

The two things we would most like to know at any time are the native and engineered erythrocyte concentrations and the concentration of HIV virions. We would also like to know the concentration of T-cells and other cells that HIV infects.

The number of engineered RBCs depends on the birth rate and the death rate. The birth rate is dependent on the number of progenitors that we add to the body and the differentiation rate into the final product. The death rate is dependent on the rate at which the spleen filters the engineered RBCs out of the bloodstream.

The number of HIV also has a number of variables factoring into the birth and death rates. The birth rate depends on the rate of infection of T-cells, the T-cell population, and the rate at which HIV becomes latent in the host cell. The death rate depends on the rate at which HIV naturally becomes inviable and the ratio of the infection of RBCs relative to T-cells.

Courtney
Before we inject our treatment into an animal or person, we are going to want to perform in vitro tests.
 * [Slide – In Vitro Tests]

To confirm that our engineered cells are expressing what we want to express, we will do genomic testing on the hematopoietic stem cells and proteomic testing on the differentiated erythrocytes. Next, we will make sure the red blood cells effectively fight off infection. Finally, we’ll find the ideal ratio of engineered erythrocyte to T cell by performing co-culture experiments.

Add picture with petri dish of cells.

Jessie

 * [Slide - In Vivo Tests]

Following the various in vitro assays, we will need to conduct in vivo experiments in animal models before we can proceed onto clinical trials.

The key points we'll be looking at are:
 * 1) Is the treatment safe in vivo? Are there any off target effects?
 * 2) Does the treatment lower viral titer? By how much?
 * 3) What is the optimal number of Hematopoietic Stem Cells we need to inject?

One animal model that could potentially work very well is the pig-tailed macaque. Not only do these macaques have HSCs that are similar to those of humans, the animals can also be infected by a human-simian hybrid virus that has a 95% similarity with HIV-1.

Hatziioannou pmid=19255423

David
-The ‘holy shit someone already patented it’ needs a better spin (emphasize how we’re doing it differently by injecting the stem cells)

I’m going to talk about the intellectual property issues surrounding our idea of a red blood cell HIV trap. There are basically two patents that may be relevant to our design, here is the first one.
 * [Slide - IP]

The 1997 patent dealt with red blood cells engineered to have CD4 on their surface. While this may seem similar to our idea, their idea revolved around manually inserting CD4 into the membrane, and that differs from our idea which uses genetic engineering to introduce CD4 into red blood cells. Their patent also differs in its proposed mechanism. They wanted their red blood cells to fuse with HIV-infected cells to form what are called syncytia, cells with multiple nuclei. The red blood cell cytotoxic load is then released into the infected cells, killing both cells.

The second patent, which I’ll call the Glaser patent, is much closer to our idea. It was issued just a few months ago, to a sole inventor who we do not believe is linked to any company. His patent covers the use of red blood cells genetically modified with CD4 and coreceptors to entrap HIV virions. One difference between our idea and this patent is that in our design, we plan to inject hematopoietic stem cells into patients, which will then differentiate into red blood cells, whereas the Glaser patent specifies directly injecting red blood cells. As far as we know, Mr. Glaser has not exclusively licensed his patent to anyone, so if we were to go forward and create a company with this idea, we would probably have to license this patent from him.
 * [Slide - Glaser patent]


 * [Slide - Summary]- unless someone wants me to write it out, I don't think I'm gonna write out what to say for the summary slide...yea