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. 2017 Mar 29:5:76-82.
doi: 10.1016/j.omtm.2017.03.005. eCollection 2017 Jun 16.

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

Xiaomei Wang  1 , Roland W Herzog  1 , Barry J Byrne  2 , Sandeep R P Kumar  1 , Qi Zhou  3 , Christian J Buchholz  3 , Moanaro Biswas  1
Affiliations

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

Xiaomei Wang et al. Mol Ther Methods Clin Dev. .

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.

Keywords: CD20; SCFV; gene transfer; hemophilia B; lentivirus; measles virus; psuedotype.

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Figures

Figure 1
Figure 1
Retargeting LVs with Specificity for Human CD20 Expressing B Cells (A) Schematic representation of the lentiviral construct expressing the IgG-hFIX fusion protein. LTR, long terminal repeat; ψ, packaging element; SFFVp, spleen focus forming virus promoter; eGFP, enhanced green fluorescent protein; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; 3′ SIN LTR, self-inactivating at the U3 region of the 3′ LTR. (B) Representative cartoon depicting the principle of CD20-LV. As compared to VSV-LV, CD20-LVs are pseudotyped with the MV H and F envelope glycoproteins. Retargeting is achieved by mutating surface receptor binding sites on the H protein (Hmut) to abolish binding to the natural receptors SLAM and CD46. Addition of scFv, consisting of the heavy and light elements of the variable region of an Ig molecule specific for hCD20 connected by a linker, redirects receptor specificity to hCD20-expressing cells.
Figure 2
Figure 2
Representative Histogram and Intensity Dot Plots Indicating Transduction Efficacies Achieved with CD20-LVGFP at Different MOIs Primary human and hCD20 transgenic mouse B cells were transduced at 6, 1, and 0.3 MOI of LV, and percent GFP+ cells were quantified after 7-AAD exclusion of dead cells. Histogram overlay at 6 MOI (black histogram: untransduced cells, red histogram: LV-transduced cells) indicate the intensity of GFP expression. Data are representative of at least two experiments.
Figure 3
Figure 3
CD20-LVGFP Does Not Transduce WT Mouse B Cells (A) Representative histogram and intensity dot plots showing negligible transduction (% GFP+) of WT BALB/c B cells by CD20-LVGFP at an MOI of 6. (B) Comparison of transduction efficacies of hCD20-tg mouse B cells and WT BALB/c B cells (% GFP+) by CD20-LVGFP at an MOI of 1. Data are representative of at least two experiments.
Figure 4
Figure 4
Adoptive Transfer of CD20-LVhFIX-IgG Transduced B Cells Prevents Inhibitor Formation in Response to Adjuvant Challenge with hFIX (A) Experimental outline and timeline. BALB/c HB mice (n = 6) received CD20-LVhFIX-IgG transduced hCD20 transgenic mouse B cells at 5–10 × 106 cells/mouse. This was followed 24 hr later by SC injection of 1 IU of hFIX in adjuvant. Blood was collected on weeks 2 and 3. (B) Comparison of anti-FIX IgG1 titers (ng/mL) from control and cell transfer recipient mice. (C) Inhibitor titers (BU/mL) from control mice and mice that received transduced B cells. Data are average ± SD. Statistical differences are indicated for each time point.
Figure 5
Figure 5
CD20-LVhFIX-IgG Transduced B Cells Reduce Inhibitor Formation in Response to IV Challenge with hFIX (A) Experimental outline and timeline. BALB/c HB mice (n = 4) received CD20-LVhFIX-IgG transduced hCD20-tg mouse B cells at 5–10 × 106 cells/mouse. This was followed by eight weekly IV injections of 3 IU of hFIX. Blood was collected on week 8. (B) Comparison of anti-FIX IgG1 titers (ng/mL) from control and cell transfer recipients. (C) Inhibitor titers (BU/mL) from control mice and mice that received transduced B cells. The incidence of inhibitor formation for both groups of mice is indicated. Data are average ± SD. Statistical differences are indicated for each time point.
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