Site Specific Knock-In Genome Editing in Human HSCs Using Baboon Envelope gp Pseudotypedviral Derived “Nanoblades” Loaded with Cas9/sgRNA Combined with Donor Encoding AAV-6

Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. Here, we have designed ?Nanoblades?, a new technology that will deliver a genomic cleaving agent into cells. These are genetically modified Murine Leukemia Virus (MLV) or HIV derived virus like particle (VLP), in which the viral structural protein Gag has been fused to the Cas9. These VLPs are thus loaded with Cas9 protein together with the guide RNAs. Thus, nanoblades are devoid of any viral-derived genetic material. Highly efficient gene editing was obtained in cell lines, IPS cells and primary mouse and human cells (Mangeot et al. Nature Communication, 2019). However, their delivery into target cells can be technically challenging when working with primary immune cells. Now we showed that nanoblades were remarkably efficient for entry into human T, B and hematopoietic stem cells thanks to their surface co-pseudotyping with baboon retroviral and VSVG envelope glycoproteins. We were able to induce efficient, transient and very rapidlygenome-editing in human induced pluripotent stem cells reaching up to 70% in the empty spiracles homeobox 1 (EMX1) and muscular dystrophy (MD) gene locus. A brief nanoblade incubation of primary human T and B cells resulted in 40% and 20% editing of the Wiskott-Aldrich syndrome (WAS) gene locus, while hematopoietic stem cells treated for 18 h with nanoblades allowed 30-40% gene editing in the WAS gene locus and up to 80% for the Myd88 genomic target. Moreover, for the HIV- and MLV-derived nanoblades no cell toxicity and low to undetectable off-target effects were demonstrated.Finally, we also treated hHSCs with nanoblades in combination with an AAV-6 donor encoding vector resulting in over 20% of stable expression cassette knock-in into the WAS gene locus. Currently, we are evaluating these gene modified HSCs for their long-term reconstitution of NOD/SCIDgC-/- mice.Summarizing, this new technology is simple to implement in any laboratory, shows high flexibility for different targets including primary immune cells of murine and human origin, is relatively inexpensive and therefore have important prospects for basic and clinical translation in the area of gene therapy.Fil: Gutierrez, Alejandra. Inserm; FranciaFil: Abrey Recalde, Maria Jimena. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; Argentina. Inserm; FranciaFil: Mangeot, Philippe E.. Inserm; FranciaFil: Costa, Caroline. Inserm; FranciaFil: Bernandin, Ornellie. Inserm; FranciaFil: Fusil, Floriane. Inserm; FranciaFil: Froment, Gisèle. Inserm; FranciaFil: Martin, Francisco. Inserm; FranciaFil: Bellabdelah, Karim. Universidad de Granada; EspañaFil: Ricci, Emiliano P.. Inserm; FranciaFil: Ayuso, Eduard. Universite de Nantes; FranciaFil: Cosset, François loic. Inserm; FranciaFil: Verhoeyen, Els. Inserm; FranciaAmerican Society of Cell and Gene Therapy 22nd Annual MettingWashingtonEstados UnidosAmerican Society of Cell and Gene Therap

CD20 and CD19 targeted vectors induce minimal activation of resting B lymphocytes

B lymphocytes are an important cell population of the immune system. However, until recently it was not possible to transduce resting B lymphocytes with retro- or lentiviral vectors, making them unsusceptible for genetic manipulations by these vectors. Lately, we demonstrated that lentiviral vectors pseudotyped with modified measles virus (MV) glycoproteins hemagglutinin, responsible for receptor recognition, and fusion protein were able to overcome this transduction block. They use either the natural MV receptors, CD46 and signaling lymphocyte activation molecule (SLAM), for cell entry (MV-LV) or the vector particles were further modified to selectively enter via the CD20 molecule, which is exclusively expressed on B lymphocytes (CD20-LV). It has been shown previously that transduction by MV-LV does not induce B lymphocyte activation. However, if this is also true for CD20-LV is still unknown. Here, we generated a vector specific for another B lymphocyte marker, CD19, and compared its ability to transduce resting B lymphocytes with CD20-LV. The vector (CD19ds-LV) was able to stably transduce unstimulated B lymphocytes, albeit with a reduced efficiency of about 10% compared to CD20-LV, which transduced about 30% of the cells. Since CD20 as well as CD19 are closely linked to the B lymphocyte activation pathway, we investigated if engagement of CD20 or CD19 molecules by the vector particles induces activating stimuli in resting B lymphocytes. Although, activation of B lymphocytes often involves calcium influx, we did not detect elevated calcium levels. However, the activation marker CD71 was substantially up-regulated upon CD20-LV transduction and most importantly, B lymphocytes transduced with CD20-LV or CD19ds-LV entered the G1b phase of cell cycle, whereas untransduced or MV-LV transduced B lymphocytes remained in G0. Hence, CD20 and CD19 targeting vectors induce activating stimuli in resting B lymphocytes, which most likely renders them susceptible for lentiviral vector transduction

Cell cycle progression of resting primary human B lymphocytes after transduction with targeted vectors.^1.

1<p>Cells were analyzed 48 h after transduction.</p>2<p>Unstimulated B lymphocytes were analyzed.</p>3<p>B lymphocytes were stimulated for 48 h with a cytokine cocktail consisting of 50 ng/ml IL-2, 300 ng/ml CD40Ligand, 10 ng/ml IL-4 and 10 ng/ml IL-10.</p>4<p>not applicable.</p>5<p>not done.</p

Stable transduction of unstimulated primary human B lymphocytes by CD20-LV and CD19ds-LV.

<p>(<b>A</b>) Freshly isolated primary human B lymphocytes from six different donors were transduced with CD20-LV, CD19ds-LV or MV-LV at an MOI of 2, or VSVG-LV at an MOI of 100. Forty-eight hours later, B lymphocytes were identified by their CD20 expression and the percentage of EGFP⁺ cells in the CD20-gated cells was determined by FACS analysis. Results are expressed as mean ± SEM. *, ** and *** indicate <i>P</i><0.05, <i>P</i><0.01 and <i>P</i><0.001 versus transduction with CD20-LV, respectively. Data were analyzed by one-way ANOVA. (<b>B–C</b>) To verify stable integration of the <i>egfp</i> gene into the B lymphocyte genome, freshly isolated primary human B lymphocytes (purity was ∼99.8%) from three different donors were transduced with the indicated vectors. Transduced cells were cultivated in presence of 10 ng/ml rhIL-15 and 10 ng/ml rhIL-2 for 10 days on MS-5 feeder cells. The percentage of CD19⁺/EGFP⁺ cells in the CD20-gated cells was determined by FACS analysis at the indicated time points. (<b>B</b>) FACS blots of one representative experiment are shown (at least 2000 B lymphocytes were gated for analysis). (<b>C</b>) Diagram showing a summary of all three independent experiments. Dark gray column: CD20-LV; light gray column: CD19ds-LV; white column: VSVG-LV. Results are expressed as mean ± SEM. * and ** indicate <i>P</i><0.05 and <i>P</i><0.01 for CD20-LV and CD19ds-LV versus VSVG-LV, respectively. Data were analyzed by Student’s t-test without adjustment of multiple comparisons.</p

CD20-LV and CD19ds-LV induce minimal activation of resting primary human B lymphocytes.

<p>(<b>A</b>) Freshly isolated primary human B lymphocytes were left untransduced or were incubated with CD20-LV, CD19ds-LV or VSVG-LV at an MOI of 1.5–2 or with CD20-VLP at the same HIV p24 value as CD20-LV. As positive control, B lymphocytes were activated with a cytokine cocktail consisting of 50 ng/ml IL-2, 300 ng/ml CD40Ligand, 10 ng/ml IL-4 and 10 ng/ml IL-10. Forty-eight hours later, the cell surface expression of the activation markers CD69 and CD71 was determined by FACS analysis using appropriate antibodies. Mean values of CD69⁺ and CD71⁺ cells, respectively, of four different donors and SEM are shown. *, ***, and ****indicate <i>P</i><0.05, <i>P</i><0.001 and <i>P</i><0.0001 versus untransduced cells, respectively. Data were analyzed by two-way ANOVA. (<b>B</b>) Freshly isolated primary human B lymphocytes were transduced by CD20-LV (MOI 3), CD19ds-LV (MOI 3) or MV-LV (MOI 5) vectors or remained untransduced. As positive control, B lymphocytes were activated with the same cytokine cocktail as described for (A). Forty-eight hours later, cell cycle progression was monitored by simultaneously visualizing the RNA (pyronin-Y) and DNA (7-AAD) content of the B lymphocytes by FACS analysis. The percentages of cells in G0, G1b or S/G2/M phase of the cell cycle are indicated. One representative experiment out of four independent experiments is shown.</p

CD20-VLP and CD19ds-VLP do not induce calcium influx into unstimulated primary human B lymphocytes.

<p>Freshly isolated primary human B lymphocytes were labeled with Calcium Sensor Dye eFluor® 514 and labeled cells were assessed by flow cytometry to establish a baseline level of fluorescence. Then cells were removed, incubated with the indicated stimulants, and replaced immediately for further flow cytometric analysis. The transient increase in intracellular calcium concentration was recorded by monitoring the change in MFI of the cells. To determine the background auto-fluorescence of the cells, one sample was recorded without the addition of any stimulant after baseline monitoring (mock). The gap in the histogram reflects the time-period when the tube containing the cells was removed from the instrument to add the stimulants. The arrow indicates the time-point of addition of the stimulant. VLP: virus like particles; MFI: mean fluorescence intensity.</p

Evidence for Protection against Chronic Hepatitis C Virus Infection in Chimpanzees by Immunization with Replicating Recombinant Vaccinia Virus▿

Given the failures of nonreplicating vaccines against chronic hepatitis C virus (HCV) infection, we hypothesized that a replicating viral vector may provide protective immunity. Four chimpanzees were immunized transdermally twice with recombinant vaccinia viruses (rVV) expressing HCV genes. After challenge with 24 50% chimpanzee infective doses of homologous HCV, the two control animals that had received only the parental VV developed chronic HCV infection. All four immunized animals resolved HCV infection. The difference in the rate of chronicity between the immunized and the control animals was close to statistical significance (P = 0.067). Immunized animals developed vigorous gamma interferon enzyme-linked immunospot responses and moderate proliferative responses. To investigate cross-genotype protection, the immunized recovered chimpanzees were challenged with a pool of six major HCV genotypes. During the acute phase after the multigenotype challenge, all animals had high-titer viremia in which genotype 4 dominated (87%), followed by genotype 5 (13%). However, after fluctuating low-level viremia, the viremia finally turned negative or persisted at very low levels. This study suggests the potential efficacy of replicating recombinant vaccinia virus-based immunization against chronic HCV infection