Immune Modulatory Cell Therapy for Hemophilia B Based on CD20-Targeted Lentiviral Gene Transfer to Primary B Cells

Abstract

Gene-modified B cells expressing immunoglobulin G (IgG) fusion proteins have been shown to induce tolerance in several autoimmune and other disease models. However, lack of a vector suitable for gene transfer to human B cells has been an obstacle for translation of this approach. To overcome this hurdle, we developed an IgG-human factor IX (hFIX) lentiviral fusion construct that was targeted to specifically transduce cells expressing human CD20 (hCD20). Receptor-specific retargeting by mutating envelope glycoproteins of measles virus (MV)-lentiviral vector (LV) and addition of a single-chain variable fragment specific for hCD20 resulted in gene delivery into primary human and transgenic hCD20 mouse B cells with high specificity. Notably, this protocol neither required nor induced activation of the B cells, as confirmed by minimal activation of inflammatory cytokines. Using this strategy, we were able to demonstrate induction of humoral tolerance, resulting in suppression of antibody formation against hFIX in a mouse model of hemophilia B (HB). In conclusion, transduction of receptor-specific retargeted LV into resting B cells is a promising method to develop B cell therapies for antigen-specific tolerance induction in human disease.

Transient Retrovirus-Based CRISPR/Cas9 All-in-One Particles for Efficient, Targeted Gene Knockout

“… 28 , 81 , 82 The possibility to use novel pseudotyping glycoproteins makes it possible to confine delivery of our chimeric Gag.MS2 particles to specific cell types, as described for retroviral targeting strategies by Buchholz and co-workers. 83 , 84 , 85 Calculation of amounts of Cas9 (in either mRNA or protein form) and guide RNA administered for gene editing in recently published articles led to estimates in the range of 1–15 pg mRNA/cell (i.e., 4.2 × 10 5 –6.3 × 10 6 mRNA molecules/cell), 5–75 pg Cas9 protein/cell (i.e., 1.8 × 10 7 –2.8 × 10 8 protein molecules/cell), and 1–100 pg guide RNA/cell (i.e., 1.9 × 10 7 –1.9 × 10 10 guide RNA molecules/cell). 21 , 22 , 25 , 68 , 86 Based on data presented in Table 1 and an average RFP657 knockout efficiency of 44%–56%, we calculated that 9.6 × 10 2 –3.8 × 10 3 SpCas9.TS mRNA molecules/cell and 7.1 × 10 4 –5.8 × 10 5 sgRNAs/cell were delivered (to 1 × 10 5 cells with 50 μL of our 100-fold concentrated Gag.MS2 particles).…”

Surface-Engineered Lentiviral Vectors for Selective Gene Transfer into Subtypes of Lymphocytes

“…Important progress toward clinical translation of the CD20-targeted MV-LV was recently achieved by Wang and colleagues. 87 In this interesting study, B cells were isolated from human CD20-transgenic BALB/c mice and used as target cells for genetic modification by CD20-MV-LV to induce tolerance in a mouse model of hemophilia B. Astonishingly, the authors could show that CD20-MV-LV successfully transduced CD20 + mouse B cells, but not CD20 − cells, at similar efficiency as human primary B cells even without prior activation. To evaluate the clinical potential, CD20-transgenic mouse B cells were ex vivo transduced with CD20-MV-LV encoding human FIX, followed by adoptive transfer into recipient mice.…”

Innovative Approaches for Immune Tolerance to Factor VIII in the Treatment of Hemophilia A

“…However, B cells also play a role in antigen presentation ( 64 ). Interestingly, gene-modified primary B cells have the capacity to induce immune tolerance upon retroviral or lentiviral gene transfer (while TLR9 activation during plasmid gene transfer generates immunogenic B cells) ( 65 – 67 ). Skupsky et al showed that the expression of IgG fusion proteins (IgG-A2 and IgG-C2 domains of FVIII) in primary B cells is a particularly powerful tool to induce tolerance ( 65 ).…”

“…A major limitation of this approach had been a lack of suitable gene transfer vectors for human B cells. Recent development of lentiviral vectors (LV) targeted to human CD20 through inclusion of a single chain antibody fragment in the viral envelope protein has overcome this bottle neck ( 67 ). Primary B cells transduced with such a LV to express IgG-FIX prevented inhibitor formation in hemophilia B mice ( 67 ).…”

“…Recent development of lentiviral vectors (LV) targeted to human CD20 through inclusion of a single chain antibody fragment in the viral envelope protein has overcome this bottle neck ( 67 ). Primary B cells transduced with such a LV to express IgG-FIX prevented inhibitor formation in hemophilia B mice ( 67 ). However, clinical translation is still not straightforward because of relatively low titers of this vector.…”