The authors want to acknowledge the staff of the flow cytometry facility and animal facility of CSCR. A. C. is funded by an ICMR-SRF fellowship, K. V. K. is funded by a DST-INSPIRE fellowship, and P. B. is funded by a CSIR-JRF fellowship. This work was funded by the Department of Biotechnology, Government of India (grant no. BT/PR26901/MED/31/377/2017 and BT/PR31616/MED/31/408/2019)

In vivo experiments on immunodeficient mice were approved by and performed following the guidelines of the Institute Animal Ethics Committee (IAEC), Christian Medical College, Vellore, India. Granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood samples were collected from healthy human donors with informed consent after obtaining Institutional Review Board (IRB) approval.
1. Isolation of peripheral blood mononuclear cells (PBMNCs) and purification of CD34 <su...

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The authors declare that no competing financial interests exist.

The successful outcome of HSPC gene therapy relies predominantly on the quality and quantity of engraftable HSCs in the graft. However, the functional properties of HSCs are highly affected during the preparatory phase of gene therapy products, including by in vitro culture and toxicity associated with the gene manipulation procedure. To overcome these limitations, we have identified ideal HSPCs culture conditions that retain the stemness of CD34+CD133+CD90+ HSCs in ex&#160;v...

The inaccessibility to human leukocyte antigen (HLA)-matched donors in allogenic transplantation settings and the rapid development of highly versatile and safe genetic engineering tools make autologous hematopoietic stem cell transplantation (HSCT) a curative treatment strategy for the hereditary blood disorders1,2. Autologous hematopoietic stem and progenitor cell (HSPC) gene therapy involves the collection of patients&#39; HSPCs, genetic manipulation, correction of disease-causing mutations, and transplantation of gene-corrected HSPCs into the patient3,4. However, the successful outcome of the gene therapy relies on the quality of the transplantable gene-modified graft. The gene manipulation steps and ex vivo culture of HSPCs affect the quality of the graft by decreasing the frequency of long-term hematopoietic stem cells (LT-HSCs), necessitating the infusion of large doses of gene-manipulated HSPCs2,5,6.
Several small molecules, including SR1 and UM171, are currently being employed to expand cord blood HSPCs robustly7,8. For adult HSPCs, due to the higher cell yield obtained on mobilization, robust expansion is not required. However, retaining the stemness of isolated HSPCs in ex vivo culture is crucial for its gene therapy applications. Therefore, an approach focusing on the culture enrichment of hematopoietic stem cells (HSCs) is developed using a combination of small molecules: Resveratrol, UM729, and SR1 (RUS)7. The optimized HSPC culture conditions promote the enrichment of HSCs, resulting in increased frequency of gene-modified HSCs in vivo, and reduce the need for gene manipulating large doses of HSPCs, facilitating cost-effective gene therapy approaches8.
Here, a comprehensive protocol for HSPCs culture is described, along with the infusion and analysis of gene-edited cells in vivo.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >4D-Nucleofector&#174; X Unit</td>
			<td >LONZA BIOSCIENCE</td>
			<td >AAF-1003X</td>
			<td ></td>
		</tr>
		<tr >
			<td >4D-Nucleofector&#8482; X Kit ( 16-well Nucleocuvette&#8482; Strips)</td>
			<td >LONZA BIOSCIENCE</td>
			<td >V4XP-3032</td>
			<td></td>
		</tr>
		<tr >
			<td >Antibiotic-Antimycotic (100X)</td>
			<td >THERMO SCIENTIFIC</td>
			<td >15240096</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-human CD45 APC</td>
			<td >BD BIOSCIENCE&#160;</td>
			<td >555485&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-human CD13 PE</td>
			<td >BD BIOSCIENCE</td>
			<td >555394</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-human CD19 PerCP</td>
			<td >BD BIOSCIENCE</td>
			<td >340421</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-human CD3 PE-Cy7</td>
			<td >BD BIOSCIENCE</td>
			<td >557749</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-human CD90 APC</td>
			<td >BD BIOSCIENCE</td>
			<td>561971</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-human CD133/1&#160;</td>
			<td >Miltenyibiotec</td>
			<td >130-113-673</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-human CD34 PE</td>
			<td >BD BIOSCIENCE</td>
			<td >348057</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-mouse CD45.1 PerCP-Cy5</td>
			<td >BD BIOSCIENCE</td>
			<td >560580</td>
			<td></td>
		</tr>
		<tr >
			<td >Blood Irradator-2000</td>
			<td >&#160;BRIT (Department of Biotechnology, India)</td>
			<td >BI 2000&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >Cell culture dish (delta surface-treated 6-well plates)</td>
			<td >NUNC (THERMO SCIENTIFIC)</td>
			<td >140675</td>
			<td></td>
		</tr>
		<tr >
			<td >CrysoStor CS10</td>
			<td >BioLife solutions</td>
			<td >#07952</td>
			<td></td>
		</tr>
		<tr >
			<td >Busulfan</td>
			<td >CELON LABS (60mg/10mL)</td>
			<td >-&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >Guide-it Recombinant Cas9</td>
			<td >TAKARA BIO</td>
			<td >632640</td>
			<td></td>
		</tr>
		<tr >
			<td >Cas9-eGFP</td>
			<td >SIGMA</td>
			<td >C120040&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >&#160;Centrifuge tube-15ml</td>
			<td >CORNING</td>
			<td >430790</td>
			<td></td>
		</tr>
		<tr >
			<td >&#160;Centrifuge tube-50ml</td>
			<td >NUNC (THERMO SCIENTIFIC)</td>
			<td >339652</td>
			<td></td>
		</tr>
		<tr >
			<td >DMSO</td>
			<td >MPBIO</td>
			<td >219605590</td>
			<td></td>
		</tr>
		<tr >
			<td >DNAase</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >6469</td>
			<td></td>
		</tr>
		<tr >
			<td >Dulbecco&#8242;s Phosphate Buffered Saline- 1X</td>
			<td >HYCLONE</td>
			<td >SH30028.02</td>
			<td></td>
		</tr>
		<tr >
			<td >EasySep&#8482; Human CD34 Positive Selection Kit II</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td>17856</td>
			<td></td>
		</tr>
		<tr >
			<td >EasySep magnet</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >18000</td>
			<td></td>
		</tr>
		<tr >
			<td >Electrophoresis unit</td>
			<td >ORANGE INDIA</td>
			<td >HDS0036</td>
			<td></td>
		</tr>
		<tr >
			<td >FBS</td>
			<td >THERMO SCIENTIFIC</td>
			<td >10270106</td>
			<td></td>
		</tr>
		<tr >
			<td >Flow cytometer &#8211; ARIA III</td>
			<td >BD BIOSCIENCE</td>
			<td >-&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >FlowJo&#160;</td>
			<td >BD BIOSCIENCE</td>
			<td >&#160;-</td>
			<td></td>
		</tr>
		<tr >
			<td >Flt3-L</td>
			<td >PEPROTECH</td>
			<td >300-19-1000</td>
			<td></td>
		</tr>
		<tr >
			<td >Gel imaging system</td>
			<td >CELL BIOSCIENCES</td>
			<td >11630453</td>
			<td></td>
		</tr>
		<tr >
			<td >HighPrep DTR reagent</td>
			<td >MAGBIOGENOMICS</td>
			<td >DT-70005</td>
			<td></td>
		</tr>
		<tr >
			<td >Human BD Fc Block</td>
			<td >BD BIOSCIENCE</td>
			<td >553141</td>
			<td></td>
		</tr>
		<tr >
			<td >IL6</td>
			<td >PEPROTECH</td>
			<td >200-06-50</td>
			<td></td>
		</tr>
		<tr >
			<td >IMDM media</td>
			<td >THERMO SCIENTIFIC</td>
			<td >12440053</td>
			<td></td>
		</tr>
		<tr >
			<td >Infrared lamp</td>
			<td >MURPHY</td>
			<td >-</td>
			<td></td>
		</tr>
		<tr >
			<td >Insulin syringe 6mm 31G</td>
			<td >BD BIOSCIENCE</td>
			<td >324903</td>
			<td></td>
		</tr>
		<tr >
			<td >Ketamine</td>
			<td >KETMIN 50</td>
			<td >-</td>
			<td></td>
		</tr>
		<tr >
			<td >Loading dye 6X</td>
			<td >TAKARA BIO</td>
			<td >9156</td>
			<td></td>
		</tr>
		<tr >
			<td >Lymphoprep</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >7851</td>
			<td></td>
		</tr>
		<tr >
			<td >Mice Restrainer</td>
			<td >AVANTOR</td>
			<td >TV-150</td>
			<td></td>
		</tr>
		<tr >
			<td >Nano drop spectrophotometer</td>
			<td >THERMO SCIENTIFIC</td>
			<td >ND-2000C</td>
			<td></td>
		</tr>
		<tr >
			<td >Neubauer cell counting chamber</td>
			<td >ROHEM INSTRUMENTS</td>
			<td >CC-3073</td>
			<td></td>
		</tr>
		<tr >
			<td >NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG)</td>
			<td >The Jackson Laboratory</td>
			<td>RRID:IMSR_JAX:005557</td>
			<td></td>
		</tr>
		<tr >
			<td >NOD,B6.SCID Il2r&#947;&#8722;/&#8722;KitW41/W41 (NBSGW)</td>
			<td >The Jackson Laboratory</td>
			<td>RRID:IMSR_JAX:026622</td>
			<td></td>
		</tr>
		<tr >
			<td >Nunc delta 6-well plate</td>
			<td >THERMO SCIENTIFIC</td>
			<td >140675</td>
			<td></td>
		</tr>
		<tr >
			<td >Polystyrene round-bottom tube</td>
			<td >BD</td>
			<td >352008</td>
			<td></td>
		</tr>
		<tr >
			<td >P3 primary cell Nucleofection solution</td>
			<td >LONZA BIOSCIENCE</td>
			<td >PBP3-02250</td>
			<td></td>
		</tr>
		<tr >
			<td >Pasteur pipette</td>
			<td >FISHER SCIENTIFIC</td>
			<td >13-678-20A</td>
			<td></td>
		</tr>
		<tr >
			<td >PCR clean-up kit</td>
			<td >TAKARA BIO</td>
			<td >740609.25</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse Pie Cage</td>
			<td>FISCHER SCIENTIFIC</td>
			<td>50-195-5140</td>
			<td></td>
		</tr>
		<tr >
			<td >polystyrene round-bottom tube (12 x 75 mm)</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >38007</td>
			<td></td>
		</tr>
		<tr >
			<td >Primer3</td>
			<td >Whitehead Institute for Biomedical Research</td>
			<td >https://primer3.ut.ee/</td>
			<td></td>
		</tr>
		<tr >
			<td >QuickExtract&#8482; DNA Extraction Solution</td>
			<td >Lucigen</td>
			<td >QE09050</td>
			<td></td>
		</tr>
		<tr >
			<td >Reserveratrol</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >72862</td>
			<td></td>
		</tr>
		<tr >
			<td >SCF</td>
			<td >PEPROTECH</td>
			<td >300-07-1000</td>
			<td></td>
		</tr>
		<tr >
			<td >SFEM-II</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >9655</td>
			<td></td>
		</tr>
		<tr >
			<td >sgRNA</td>
			<td >SYNTHEGO</td>
			<td >-&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >SPINWIN</td>
			<td >TARSON</td>
			<td >1020</td>
			<td></td>
		</tr>
		<tr >
			<td >StemReginin 1</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >72342</td>
			<td></td>
		</tr>
		<tr >
			<td >ICE analysis tool</td>
			<td >SYNTHEGO</td>
			<td >https://ice.synthego.com/</td>
			<td></td>
		</tr>
		<tr >
			<td >Tris-EDTA buffer solution (TE) 1X</td>
			<td >SYNTHEGO</td>
			<td >Supplied with gRNA&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >Thermocycler</td>
			<td >APPLIED BIOSYSTEMS</td>
			<td >4375305</td>
			<td></td>
		</tr>
		<tr >
			<td >TPO</td>
			<td >PEPROTECH</td>
			<td >300-18-1000</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypan blue</td>
			<td >HIMEDIA LABS</td>
			<td >TCL046</td>
			<td></td>
		</tr>
		<tr >
			<td >UM171</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >72914</td>
			<td></td>
		</tr>
		<tr >
			<td >UM729</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >72332</td>
			<td></td>
		</tr>
		<tr >
			<td >Xylazine</td>
			<td >XYLAXIN - INDIAN IMMUNOLOGICALS LIMITED</td>
			<td >-</td>
			<td></td>
		</tr>
	</tbody>
</table>

crispr/cas9 gene editing of hematopoietic stem and progenitor cells for gene therapy applications

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.

The present study identifies ideal HSPC culture conditions that facilitate the retention of CD34+CD133+CD90+ HSCs in ex vivo culture. To demonstrate the culture enrichment of HSCs along with the enhanced generation of gene-modified HSCs, the optimized procedures for PBMNC isolation, CD34+ cell purification, culture, gene editing, transplantation, characterization of engraftment, and gene-modified cells in vivo are provided (Figure 1</stron...

Watch this Scientific Journal Video about CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications at JoVE.com

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

The present protocol describes an optimized hematopoietic stem and progenitor cell (HSPC) culture procedure for the robust engraftment of gene-edited cells in vivo.

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene ...

Ex vivo culture can hamper the repopulation potential of hematopoietic stem and progenitor cells. Our protocol preserves the stemness during the culture and thus improves the frequency of gene-modified cells in vivo. Hematopoietic stem cell gene therapy is currently being employed for monogenic diseases and infectious disease like HIV.The HSPC gene therapy protocol heavily relies on ex vivo culture. Our protocol should be compatible with any procedure that employs ex vivo culture. Demonstrating this protocol will be Vigneshwaran Venkatesan, PhD student from my laboratory.After isolating the HSPCs, culture them in stem cell culture media that contains cytokines and small molecules Resveratrol, UM729, and StemRegenin1. Before gene editing, set the ThermoMixer at 37 degrees Celsius to reconstitute the guide RNA, then preheat the TE buffer at 37 degrees Celsius for 10 minutes. Centrifuge the synthetic chemically-modified sgRNA vial at 11, 000 times g for 1 minute at 4 degrees Celsius and add 15 microliters of TE buffer to the vial containing 1.5 nanomolar of lyophilized sgRNA and mix by gentle swirling to get a final concentration of 100 picomolar per microliter.Incubate the vial in the ThermoMixer with minimal shaking at 37 degrees Celsius for 30 to 40 seconds. Gently tap the vial on the sides and spin the vial to collect 15 microliters of sgRNA. Make 1-microliter aliquots and store them at minus 80 degrees Celsius for up to 1 year.For nucleofection, pellet 2 times 10 to the 5th cells by centrifuging at 200 times g for 5 minutes at room temperature. Once the supernatant is discarded, resuspend the pellet in 20 microliters of the buffer. Gently mix the cell suspension with the prepared RNP complex avoiding air bubbles.Now, transfer the suspension to the commercial nucleofection strip and electroporate the cells using the 4D-Nucleofector by selecting the pulse code DZ-100. To the electroporated cells in the nucleofection strip, add 100 microliters of pre-incubated culture media and leave the cells undisturbed for 10 minutes at room temperature. At the end of the incubation, transfer the contents to the culture plate as per the experimental requirements.Place the NSG mice in the pie cages in 6 to 8 hours prior to HSPC transplantation. Irradiate the mice at 3.5 gray using a commercially-available irradiator. For NBSGW, pre-condition 6 to 8-week-old male and female mice by intraperitoneally injecting busulfan at a dose of 12.5 milligrams per kilogram of body weight 48 hours before HSPC transplantation.For infusing 1 mouse, pellet 6 times 10 to the 5th cells in a 1.5-milliliter tube by centrifuging at 200 times g for 5 minutes. at room temperature. Gently pipette out the supernatant without disturbing the pellet and resuspend the cell pellet in 100 microliters of PBS.Place the pre-conditioned NSG or NBSGW mice in the mouse restrainer and infuse the HSPCs by tail vein injection. Hold the mouse tail and gently push the plug to restrain the mouse. Gently wipe the mouse tail with 70%ethanol.Using a 31 gauge insulin syringe, aspirate 100 microliters of the cell suspension. Now, direct the light from the infrared lamp onto the tail for 30 to 40 seconds, covering the body area of the mouse with folds of tissue paper. To maintain the tail in the planer axis with the syringe, lift it with the left index finger.Gently insert the bevel part of the needle into the left or right caudal tail vein at an angle of 20 degrees. Push the plunger to infuse the cell suspension into the vein and apply gentle pressure near the pierced region with tissue paper and pull out the needle. After anesthetizing, position the animal in ventral recumbency and gently scruff the mice to open the eye, allowing the eye's globe to protrude slightly.Gently insert the pasture pipette at a 30 to 45 degree angle into the medial canthus of the eye under the nictating membrane. After placing the Pasteur pipette at the proper position, apply slight pressure to the tube and begin to rotate the tube gently. After collecting 50 to 80 microliters of peripheral blood, gently withdraw the pipette from the medial canthus of the eye.Post-euthanization, make a vertical incision 1 centimeter above the urethra and extend until 1 centimeter below the diaphragm. Cut horizontally at the incised area's corners to open the abdominal region. After dissecting the femur and tibia, use scissors to remove the soft tissues attached to the femur and tibia and gently scrub the bones with a tissue paper.Use a scalpel to make a small hole with a diameter not more than 0.2 centimeters at the bottom of a 0.5-milliliter microcentrifuge tube. Remove the proximal ends of the bones using a scalpel and place the bones with the cut side facing toward the hole of the 0.5-milliliter micro centrifugation tube. Now, place the 0.5-milliliter tube with the bones in the 1.5-milliliter tube containing 100 microliters of sterile PBS.Close the lid, spin the tubes at 200 times g for 3 minutes under sterile conditions at room temperature and discard the 0.5-milliliter tubes containing bones with an empty marrow cavity. To the 1.5-milliliter reaction tube containing bone marrow, add 1 milliliter of PBS, then using a 1-milliliter pipette, gently resuspend the cells 10 times. Transfer 1 milliliter of the cell suspension to a 15-milliliter tube containing 9 milliliters of RBC lysis buffer.Incubate the cells in ice for 7 minutes by gently inverting the tube every two minutes. After incubation, centrifuge the tube at 200 times g for 5 minutes at room temperature and repeat the washing until a clear pale white pellet is observed. The frequency of live cells and CD34-positive, CD90-positive cells have increased in the RUS group after 48 hours of nucleofection.After 72 hours, the percentage and frequency of indel increased in the RUS-treated group as compared to DMSO-treated group suggesting an increase in gene editing. The absolute number of CD34-positive, CD90-positive cells, and total nucleated cells are significantly higher 48 hours post-editing. Flow cytometry analysis of human CD45-positive cells in the NSG mice showed increased engraftment in the culture conditions.Analysis of the gene-editing frequency in the mouse BM cells showed increased engraftment of gene-edited HSPCs in RUS-supplemented culture conditions. Culturing the HSPCs at a confluence of 200, 000 cells per mL is crucial to improve the functional stem cells. Amplifying the cord blood stem cells and the iSPC-derived stem cells are the new areas in which this protocol is being tested.

crisprcas9-gene-editing-hematopoietic-stem-progenitor-cells-for-gene

Research

JoVE Journal

Biology

3.2K visualizações.  Christian Medical College campus. A cultura ex vivo pode dificultar o potencial de repovoamento de células-tronco hematopoiéticas e progenitoras. Nosso protocolo preserva a caule durante a cultura e, assim, melhora a frequência de células modificadas por genes in vivo. A terapia genética com células-tronco hematopoiéticas está sendo empregada atualmente para doenças monogênicas e doenças infecciosas como o HIV.O protocolo de terapia gênica do HSPC depende fortemente da cultura ex vivo. Nosso protocolo deve ...