Newer humanized rodent models increase applicability of animal testing data to the treatment of human disease.
Like a nightmarish scene in one of those cheesy science fiction movies from the 1950s, human-animal chimeras are found everywhere nowadays. Examples of chimeras range from human ears grown on the backs of rats to goat-sheep chimeras (affectionately called geeps). The scientists who created these chimeras may have grown up watching those tacky movies and been thus inspired to push science fiction to become science fact. It’s as if Orwellian predictions have come true, and life in this brave new world will never be the same again. And if this trend continues, here is one more prediction: our four-legged ancient relatives will one day become bipeds and “stand up for themselves”, literally. (Insert laughter/groan here.)
But that’s a discussion for another time. Instead, let’s take a step back … to about 20 years ago, when there were not any good animal models to test HIV therapies. The year was 1988, and an important paper published by McCune, et al. (Science. 1988 Sep 23;241(4873):1632-9.) described what would eventually become known as the SCID-hu mouse model—the original humanized rodent model. SCID stands for severe combined immunodeficiency.
One researcher using humanized rodent models is Ramesh Akkina, DVM, PhD, professor, Department of Microbiology, Immunology and Pathology at Colorado State University, Fort Collins, Colo. Akkina is using the SCID-hu Thy/Liv mouse to evaluate potential gene therapies for HIV infection. Although SCID mice can be purchased from suppliers such as Jackson Laboratory, Bar Harbor, Me., Charles River Laboratories, Wilmington, Mass., and others, Akkina chooses to breed his own. After they are bred, human thymus and liver tissue (purchased from Advanced Bioscience Resources Inc., Alameda, Calif.) are implanted under the kidney capsule by microsurgery to create SCID-hu Thy/Liv mice. It is because these mice are immunodeficient that they accept the xenograft.
Direct to humans
“A major advantage with this in vivo system is that any data you get from SCID-hu mice is directly applicable to a human situation,” says Akkina. Injection of HIV into this thymus tissue leads to HIV-specific pathology characteristic of human infection, illustrating the power of this model. Akkina also uses SCID-hu to understand the effects of HIV infection on human hematopoiesis and to determine the efficacy of anti-HIV gene constructs in generating resistance to HIV infection. A typical experiment involves first inserting anti-HIV genes into CD34-positive hematopoietic progenitor cells. Then after about two months to allow the progenitors to develop into mature T-cells in SCID-hu, Akkina determines whether or not these transgenic T-cells that express anti-HIV genes are also resistant to HIV infection. Some of the potential gene therapy candidates that Akkina tested include ribozymes, siRNA molecules directed against HIV genes, as well as host proteins such as CXCR4 and CXCR5 coreceptors that aid in viral infection. Data from these experiments established that it is possible to introduce siRNAs in a blood-forming stem cell and derive progeny cells expressing HIV resistance.
click to enlarge
A SCID-hu Thy/Liv graft is injected with virus. (Source: Ramesh Akkina, DVM, PhD)
According to Akkina, there are currently ongoing clinical trials testing this gene therapy idea using a more combinatorial approach. It is reasoned that by using multiple anti-HIV genes rather than just one gene, one can circumvent the problem of HIV resistance. “So we are using combinatorial strategy where three different regions of the viral RNA are targeted in addition to cellular gene CCR5 utilizing ribozymes and siRNAs,” says Akkina. “And that actually has been shown to work in the SCID-hu system.”
Another researcher using the SCID-hu model to look at the effect of HIV infection on hematopoiesis is Jerome A. Zack, PhD, Department of Medicine, Division of Hematology-Oncology, Dept of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, Calif. “If interpretation is done correctly, a lot of info can be gained using this system that would be quicker and less expensive than doing similar experiments in the simian or non-human primate system.” Zack breeds all of the SCID mice he works with and does the subsequent implantation as well.
“We have looked for whether antiretroviral drugs added after T-cell depletion could allow T-cell reconstitution of the immune system. And they could in [SCID-hu],” says Zack. Here’s how that experiment was done. After inoculating the implant with HIV, the virus was allowed to completely deplete CD4-positive cells. A powerful combination of antiretroviral was then administered. Concomitantly, exogenous stem cells were added to one group of treated, infected implants while the other group did not receive these cells. “And it actually turned out that we got reconstitution in basically all of the mice,” he explains. “So that indicated to us that progenitor cells that remained in the implant after CD4 loss were still able to turn into T-cells if we added antiretrovirals, suggesting that the thymic stromal elements, as well as the progenitors, are still functional in those implants.” Zack adds that reconstitution of cells throughout an experiment is one of the advantages that the SCID-hu mouse has over cell culture systems.
In the future, “I would like to see the [SCID-hu] technology advanced to where full human immunoreconstitution and robust human immune responses can be seen. If that occurs, it becomes a very powerful small animal system to study all kinds of things such as vaccination and pathogenesis. It is beginning to move in that direction, but I think more optimization is needed,” says Zack.
A race against time
Cheryl Stoddart, PhD, has been using SCID-hu mice since 1995. But the history of SCID-hu use in her mentor Mike McCune’s lab at the University of California at San Francisco predates her tenure as an assistant professor in the Division of Experimental Medicine there. Stoddart and colleagues use the SCID-hu model to evaluate new HIV therapies and have a contract with the National Institutes of Health (NIH) to do just that. Their work begins when they purchase the CB.17 SCID mouse from Taconic Labs, Hudson, N.Y. After receiving these mice, they create the SCID-hu Thy/Liv mouse using the standard microsurgery procedure. “We can make 50 mice per group and that allows us to have seven groups of seven mice each per experiment. And none of the other humanized mouse models that I know of are capable of those kinds of numbers. And so that is one of the reasons we like the Thy/Liv model,” says Stoddart.
Eighteen weeks subsequent to thymus tissue implant, a thymus replete with T-cells is formed and is inoculated with HIV. Over the next three weeks, three different doses of a “test” HIV therapeutic agent (one per group) or a placebo are administered by gavage to four different groups of SCID-hu mice. Three weeks later, the mice are sacrificed, the implant removed, and HIV viral load determined. “In order to show bona fide potent antiviral activity, you like to see a greater than one log reduction in the treated groups compared to the untreated groups,” says Stoddart. She adds that one major benefit of using the SCID-hu mouse for this experiment is that the virus does not escape the human tissue and cause a systemic infection in the mouse.
But SCID-hu only roughly models how to treat HIV infection in humans. “With mice, we have the luxury of giving as much drug as we want without causing toxicity, says Stoddart. “And when you do the conversion, mice have a different body weight to surface area ratio. In order to take that into consideration, you typically have to give mice 12 times more drug on a per kilogram basis to achieve roughly the same plasma drug levels, assuming that the pharmacokinetics are the same in mice and humans.”
One thing that Stoddart and colleagues struggle with is the fact that, due to their immunodeficiency, SCID mice develop thymic lymphoma when they reach about eight months of age. And this fact obviously shortens the amount of time they can collect data on these mice, making Stoddart feel like she is in “a race against time to get the data before the mice begin to die.”
SCID, no hu
Lidia Sambucetti, PhD, occasionally uses the SCID mouse for her work at the Cancer Research Center, SRI International, Menlo Park, Calif. In her case, the typical experimental design generally involves implanting different human cancer cells into a location in the mouse and then allowing those cells to form a tumor. For other experiments, they inject the cells systemically and then determine where they lodge to form tumors or form metastases. And still other times the cells might be injected orthotopically into a certain organ and then tracked for tumor growth or metastasis. According to Sambucetti, there is not as much rejection of these tumors in the SCID mice as in the other rodent models they use. So, in the SCID mouse, these cells grow and spread usually three to four weeks after a tumor is implanted or injected, whereas in some of the less immunodeficient models, this process can take months.
Following introduction of cancer cells into these mice, Sambucetti and colleagues track the tumors in a number of different ways, depending on the tumor’s location. If the tumor is near the surface, such as a subcutaneous or mammary tumor, they are able to observe the tumor forming under the skin and simply measure its growth over time with a caliper. In contrast, internal tumors such as those in the prostate are more difficult to measure. For these tumors, Sambucetti and colleagues begin the process by injecting bioluminescent tumor cells into the prostate gland in SCID mice. The amount of bioluminescence (light) produced is a reflection of the number of cells present in the tumor. A typical design might involve measuring the effect of an experimental cancer agent on tumor growth; the ideal result here would be a reduction in tumor growth over time in the treatment group compared to the placebo group.
In summary, SCID mice have been used as animal models for almost 30 years. And for 20 years, human tissue has been transplanted into these mice to study immune function, hematopoiesis, HIV infection, and to test the potential for novel gene therapies. Luckily for researchers, the SCID-hu mouse, the first humanized rodent model, has plenty of company, including RAG-hu (see sidebar). But SCID-hu remains a powerful small animal model and continues to improve.
This article was published in Drug Discovery & Development magazine: Vol. 11, No. 6, June, 2008, pp. 16-20.
Filed Under: Drug Discovery