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

Summary

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

Abstract

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_γ^-/-)-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.

Introduction

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 treatment^1-3. 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....

Protocol

Ethic Statement: 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 the guidelines in The Guide for the Care and Use of Laboratory Animals of the National Research Council and the accreditation and guidelines of t....

Results

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 CD4ζCAR. The CD4ζCAR used in this protocol contains CD4 chimeric antigen receptor and GFP that can be.......

Discussion

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,.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CD34 microbead kit	Miltenyi	130-046-702	For sorting human CD34+ progenitor cells
Bambanker	Wako	302-14681	For freezing cells
QIAamp Viral RNA kit	Qiagen	52904	For measuring viral load in the serum
MACSQuant Flow Cytometer	Miltenyi		For flow analysis
BD LSRFortessa™	BD biosciences		For flow analysis
Hyaluronidase	Sigma	H6254-500MG	For tissue digestion
Deoxyribonuclease I	Worthington	LS002006	for tissue digestion
Collagenase	Life technology	17104-019	for tissue digestion
CFX Real time PCR detection system	Biorad		For measuring viral load and gene expression
Mice, strain NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ	The Jackson Laboratory	5557	For constructing the humanized mice
Penicillin Streptomycin (Pen Strep)	Thermo Fisher Scientific	10378016	For culturing cells
Piperacillin/tazobactam	Pfizer	Zosyn	Anti-fungal
Amphotericin B (Fungizone antimycotic)	Thermo Fisher Scientific	15290-018	Anti-fungal
Autoclip Wound Clips, 9 mm - 1,000 units	Becton Dickinson	427631	For surgery
Sterile Poly-Reinforced Aurora Surgical Gowns, 30 per case	Medline	DYNJP2707	For surgery
Sutures, 4-0, vicryl	Owens and Minor	23000J304H	For surgery
Alcohol prep pads	Owens and Minor	3583006818	For surgery
Gloves, surgical, 6 1/2	Owens and Minor	4075711102	For surgery
Yssel’s Serum-Free T-Cell Medium	Gemini Bio-products	400-102	For CD34+ cell transduction
Human Serum Albumin	Sigma-Aldrich	A9511	For CD34+ cell transduction