Name: dextran enhances the lentiviral transduction efficiency of murine and human primary nk cells
Uploaded: 2018-01-15T12:30:00
Description: Watch this Scientific Journal Video about Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells at JoVE.com

We thank Lucia Sammarco and her Lulu&#39;s Lemonade Stand for inspiration, motivation, and support. This work was supported in part by NIH R01 AI102893 and NCI R01 CA179363 (S.M.); NHLBI-HL087951 (S.R.); NIH-CA151893-K08 (M.J.R.); NCI 1R01CA164225 (L.W); the Alex Lemonade Stand Foundation (S.M.); the HRHM Program of the MACC Fund (S.M.; S.R.; M.S.T); the Nicholas Family Foundation (S.M.); the Gardetto Family (S.M.); the Hyundai Scholars Program (M.S.T.); Hyundai Hope on Wheels (S.R.); the MACC Fund (M.S.T. and S.M.); the Children&#39;s Research Institute, MCW (S.R.); and the Kathy Duffey Fogerty Award (M.J.R.).

All animal protocols followed the humane and ethical treatment of animals and were approved by the Institutional Animal Care and Use Committee (IACUC) within the Biomedical Research Center (BRC) of the Medical College of Wisconsin (MCW), Milwaukee, WI. The use of human peripheral blood mononuclear cells (PBMCs) was approved by the Institutional Review Board (IRB) of the Blood Research Institute of the Blood Center of Wisconsin, Milwaukee, WI.
1. Mice, cell lines, and vectors
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This study demonstrates that use of dextran as a cationic polymer agent enhances the lentiviral transduction efficiency of both murine and human primary NK cells. Additionally, other cationic agents, such as Pb or PS, have no discernible effect on the delivery of viral vectors into human primary NK cells. Previously, it has been demonstrated that Pb can augment gene transduction in human T cells17. These results, however, suggest that neither Pb nor PS have a similar efficiency on human primary NK...

Natural killer (NK) cells are the major lymphocytic population of the innate immune system1. NK cells function as the first-line defenders of the host immune response against tumors and infections2,3,4. NK cells also play a central role in the development of tolerance through the secretion of potent cytokines and chemokines5. Due to their potent ability to target and eliminate tumor cells, multiple clinical trials are being conducted to evaluate donor-derived human NK cells as an adoptive immunotherapy for cancer6,7. In contrast to T cells, the developmental biology of NK cells has yet to be well-characterized8. This lack of knowledge is partially due to the absence of efficient techniques that deliver genes of interest to mouse or human primary NK cells. For these reasons, most NK-cell studies have been conducted in cell lines, rather than in primary cells. Therefore, the need for a reliable and efficient protocol to transduce primary NK cells with genes of interest is crucial.
The overall goal of this study was to formulate a consistent and reliable method by which primary human or murine NK cells could be transduced with lenti- or retroviruses.
Earlier studies that attempted to address this problem have been performed, largely using the transient transformation of primary NK cells. This includes plasmid transfection9,10, Epstein-Barr Virus (EBV)/retroviral hybrid vector11, vaccinia vectors12,13, and Ad5/F35 chimeric adenoviral vectors14. Despite the modest efficiency of these techniques, the transient nature of transduction makes them unsuitable for the long-term utilization of the genetically modified NK cells. A few recent studies have used retroviral vectors to transduce NK cells, requiring multiple cycles of infection to achieve an acceptable level of gene expression11,15. In contrast to retroviral vectors, lentiviral vectors can use host-cell nuclear import machinery to translocate the viral pre-integration complex into the nucleus. This is a major limiting factor in the replication of the virus in non-dividing cells, which include primary NK cells.
Interactions between different cell-surface receptors and viral particles permit viral uptake into the cell. The initial engagements between the viral envelope proteins and their cognate host receptors could be limited because of the potential negative charges existing between these two. The rationale behind many transduction techniques is that the addition of cationic polymers, such as polybrene (Pb), protamine sulfate (PS), or dextran, could give a positive charge to the cell-surface receptors and thereby augment the binding of viral envelope proteins. This will increase the fusion efficiency and the uptake of the viral particles by the cells16. Although it has been reported that Pb or PS can improve gene transfer in T cells17, their application did not have any effect in the transduction efficiency of primary NK cells. Moreover, a comparative analysis between these reagents using primary NK cells has not been performed. In this study, the transduction efficiencies of the three cationic polymers were compared. The results show that, among these three cationic polymers, only dextran significantly enhances efficient viral transduction into both mouse and human primary NK cells.

<table>
	<tbody>
		<tr>
			<td>Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>90-64-91-9</td>
			<td></td>
		</tr>
		<tr>
			<td>polybrene (Pb)</td>
			<td>Sigma-Aldrich</td>
			<td>TR-1003</td>
			<td></td>
		</tr>
		<tr>
			<td>protamine sulfate (PS)</td>
			<td>Sigma-Aldrich</td>
			<td>p3369</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Corning</td>
			<td>25-052-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640</td>
			<td>Corning</td>
			<td>10-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ATALANTA</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>Corning</td>
			<td>30-001-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>B-mercaptoethanol</td>
			<td>SIGMA</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium pyruvate</td>
			<td>Corning</td>
			<td>MT25000CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Interferon gamma (IFN-&#947; )</td>
			<td>eBioscience</td>
			<td>14-7311-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium lodding staining solution</td>
			<td>BD</td>
			<td>51-66211E</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 3000</td>
			<td>Thermo Fisher</td>
			<td>L3000015</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>PHOENIX</td>
			<td>NDC 57319-559-05</td>
			<td></td>
		</tr>
		<tr>
			<td>NK cell negative selection kit</td>
			<td>Stem Cell</td>
			<td>19855</td>
			<td></td>
		</tr>
		<tr>
			<td>Yac-1</td>
			<td>ATCC</td>
			<td>TIB-160</td>
			<td></td>
		</tr>
		<tr>
			<td>K562</td>
			<td>ATCC</td>
			<td>CCL-243</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Jakson</td>
			<td>664</td>
			<td></td>
		</tr>
		<tr>
			<td>293T cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 flasks</td>
			<td>Cornnig</td>
			<td>430641U</td>
			<td></td>
		</tr>
		<tr>
			<td>antibody-based negative selection kits</td>
			<td>Stem Cell</td>
			<td>19055</td>
			<td></td>
		</tr>
		<tr>
			<td>51Chromium (Cr)-release assays</td>
			<td>perkin elmer&#39;s</td>
			<td>NEZ030</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA kits</td>
			<td>Ebioscience</td>
			<td>00-4201-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Butyrate</td>
			<td>Sigma</td>
			<td>5887-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear polyethylenimine</td>
			<td>polysciences</td>
			<td>23966-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll</td>
			<td>GE Life Science</td>
			<td>17-1440-03</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Corning</td>
			<td>21-022-CV</td>
			<td></td>
		</tr>
	</tbody>
</table>

dextran enhances the lentiviral transduction efficiency of murine and human primary nk cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

Dextran induces the efficient gene transfer of lentiviral vector in primary human and murine NK cells
Human NK cells were isolated and purified from PBMC (with a purity of more than 85%) and incubated overnight with rIL-2 300 U/mL. These primary NK cells were then transduced with GFP lentivirus at varied multiplicities of infection (MOI; 3, 10, and 20 IU per cell) in 24-well plates in the presence of 8 &#181;g/mL Pb, PS, or dextran...

Watch this Scientific Journal Video about Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells at JoVE.com

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

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