Name: stem-cell based engineered immunity against hiv infection in the humanized mouse model
Uploaded: 2016-07-02T13:00:00
Description: Watch this Scientific Journal Video about Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model at JoVE.com

We would like to thank Ms. Jessica Selander in providing artistic assistant in making our figures. This work was funded by grants from the NIAID/NIH, grant no. RO1AI078806, the UCLA Center for AIDS Research (CFAR), grant no. P30AI28697, the California Institute for Regenerative Medicine, grant no. TR4-06845, the American Federation for AIDS Research (amfAR), grant no. #108929-54-RGRL, and the UC Multi-campus Research Program and Initiatives, California Center for Antiviral Drug discovery (CCADD)

Ethic Statement:&#160;Human fetal tissue was obtained from Advanced Biosciences Resources or from Novogenix and was obtained without identifying information and did not require IRB approval for its use. Animal research described in this manuscript was performed under the written approval of the University of California, Los Angeles, and (UCLA) Animal Research Committee (ARC) in accordance to all federal, state, and local guidelines. Specifically, these studies were carried out under strict accordance to ...

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With CAR and HSC-based engineered immunity gaining momentum towards clinical studies, it is important to have a proper animal model to closely examine the differentiation and function of these engineered cells. In this protocol we described the methods for constructing and testing humanized mice with genetically modified stem-cells engineered against HIV. It is important to have efficient transduction of stem cells prior to transplant. However, due to the ability of T cell to proliferate upon recognition of target cells,...

Despite the success of combined anti-retroviral therapy, HIV infection is still a lifelong disease. The cellular immune response against HIV plays highly important role in controlling HIV replication. Recent advances in stem cell manipulation has allowed for the rapid development of gene therapy approaches for HIV treatment1-3. As a result, it is important to have a proper animal model that allows in vivo study of the efficacy of cell-based therapies against HIV.
Working with HIV in animal models is complicated by the fact that the virus only infects human cells. To circumvent this limitation, scientists have resorted to using disease models like the Simian Immunodeficiency Virus (SIV) in Rhesus macaques4,5. Unfortunately, there are major limitations in this model due to the inherent differences across species and the differences between SIV and HIV. Additionally, only highly specialized facilities are capable of supporting work with non-human primates and each macaque requires a large investment. Thus, there is a pressing need for a model that utilizes the human immune system, which is susceptible to HIV infection/pathogenesis, and is less financially prohibitive.
The non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-) (or NSG) Blood/Liver/Thymus (BLT) humanized mouse model is increasingly proven to be an important tool to study HIV infection. By implanting hematopoietic stem cells (HSCs) and fetal thymus, the mice are able to develop and recapitulate a human immune system1-3. One type of stem cell based gene therapy involves &#39;redirecting&#39; peripheral T cells to target HIV by reprogramming Hematopoietic Stem Cells (HSCs) to differentiate into antigen specific T cells. We have shown previously that engineering HSCs with a molecular cloned anti-HIV specific T cell receptor (TCR) against the SL9 epitope (amino acid 77-85; SLYNTVATL) of HIV-1 Gag can redirect stem cells into forming mature T cells that suppress HIV replication in the humanized NSG-BLT mouse model6. The caveat of using a molecular cloned TCR is that it is restricted to a specific human leukocyte antigen (HLA) subtype that will limit the application of this therapy. Chimeric antigen receptors (CAR), on the other hand, can be universally applied to all HLA subtypes. Initial studies were performed utilizing a CAR constructed with the extracellular and transmembrane domains of human CD4 fused to the intracellular &#950; signaling domain of CD3 (termed the CD4&#950;CAR). CD4&#950;CAR expressed on CD8 T cells can recognize HIV envelope and trigger a cytotoxic T cell response that is similar to that mediated by a T cell receptor7. We have recently demonstrated that human HSCs can be modified with CD4&#950;CAR, which can then differentiate into multiple hematopoietic lineages, including functional T cells capable of suppressing HIV replication in the humanized mouse model8. With the rapid advancement in chimeric antigen receptor therapies for cancer9, and the ongoing characterization of potent broad neutralizing antibodies10-12 against HIV that allow the construction of single chain antibody CARs, it is perceivable that many new candidate constructs, in addition to CD4&#950;CAR, will be generated and tested for stem-cell based gene therapy of HIV diseases and other diseases. In addition, the humanized NSG-BLT mouse model containing these antigen-specific CARs can also provide a useful tool to closely examine human T cell responses in vivo. Importantly, our protocol differs from previous described methods for construction of humanized of BLT mice13-15 in that the HSCs in gelatinous protein mixture is used in place of fetal liver trunks16. This protocol describes: 1) construction of humanized BLT mice engineered with CD4&#950;CAR; and 2) characterization of the differentiation of the genetically modified cells; and 3) characterization of the functionality of the genetically modified cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CD34 microbead kit</td>
			<td>miltenyi</td>
			<td>130-046-702</td>
			<td>For sorting human CD34+ progenitor cells</td>
		</tr>
		<tr>
			<td>Bambanker</td>
			<td>Wako</td>
			<td>302-14681</td>
			<td>for freezing cells</td>
		</tr>
		<tr>
			<td>QIAamp Viral RNA kit&#160;</td>
			<td>Qiagen</td>
			<td>52904</td>
			<td>For measuring viral load in the serum</td>
		</tr>
		<tr>
			<td>MACSQuant Flow Cytometer</td>
			<td>Miltenyi</td>
			<td></td>
			<td>For flow analysis</td>
		</tr>
		<tr>
			<td>BD LSRFortessa&#8482;</td>
			<td>BD biosciences</td>
			<td></td>
			<td>For flow analysis</td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma</td>
			<td>H6254-500MG&#160;</td>
			<td>For tissue digestion</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Worthington</td>
			<td>LS002006&#160;</td>
			<td>for tissue digestion</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Life technology</td>
			<td>17104-019&#160;</td>
			<td>for tissue digestion</td>
		</tr>
		<tr>
			<td>CFX Real time PCR detection system</td>
			<td>Biorad</td>
			<td></td>
			<td>For measuring viral load and gene expression</td>
		</tr>
		<tr>
			<td>Mice, strain NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ</td>
			<td>The Jackson Laboratory</td>
			<td>5557</td>
			<td>For constructing the humanized mice</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin (Pen Strep)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10378016</td>
			<td>For culturing cells</td>
		</tr>
		<tr>
			<td>piperacillin/tazobactam</td>
			<td>Pfizer</td>
			<td>Zosyn</td>
			<td>Anti-fungal</td>
		</tr>
		<tr>
			<td>Amphotericin B (Fungizone antimycotic)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15290-018</td>
			<td>Anti-fungal</td>
		</tr>
		<tr>
			<td>AUTOCLIP Wound Clips, 9 mm - 1000 units&#160;&#160;&#160;&#160;</td>
			<td>Becton Dickinson</td>
			<td>427631&#160;</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>Sterile Poly-Reinforced Aurora Surgical Gowns, 30 per case&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Medline</td>
			<td>DYNJP2707&#160;</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>sutures, 4-0, vicryl&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Owens and Minor</td>
			<td>23000J304H&#160;&#160;</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>Alcohol prep pads&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Owens and Minor</td>
			<td>3583006818</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>Gloves, surgical, 6 1/2</td>
			<td>Owens and Minor</td>
			<td>4075711102</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>Yssel&#8217;s Serum-Free T-Cell Medium</td>
			<td>Gemini Bio-products</td>
			<td>400-102</td>
			<td>For CD34+ cell transduction</td>
		</tr>
		<tr>
			<td>Human Serum Albumin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A9511</td>
			<td>For CD34+ cell transduction</td>
		</tr>
	</tbody>
</table>

stem-cell based engineered immunity against hiv infection in the humanized mouse model

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

Figure 1 shows an outline of constructing humanized BLT mice with modified stem cell. 10 weeks after the implant surgery, the mice were sacrificed to evaluate the differentiation and development of gene modified cells. As shown in Figure 2, multiple lymphoid tissues (blood, spleen, thymus and bone marrow) were harvested from a mouse that was modified with CD4&#950;CAR. The CD4&#950;CAR used in this protocol contains CD4 chimeric antigen receptor and GFP t...

Watch this Scientific Journal Video about Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model at JoVE.com

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

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