Background
B-cell proliferative disorders, such as post-transplant lymphoproliferative disease (PTLD) and certain B-cell lymphomas, occur with increased frequency among persons with T-cell compromise. These disorders are primarily Epstein–Barr Virus (EBV) + and can first present with a focal lesion accessible to direct inoculation of therapeutic agents [
4,
7]. Target tissues frequently include Waldeyer's tonsillar ring, B-cell-associated lymphoid tissues of transplanted organs, solitary lymph nodes, and central nervous system lesions. Direct introduction of oncolytic viruses into localized tumors provides theoretical advantages over chemotherapy, immunotherapy and radiation therapy by reducing systemic toxicity to which the immunocompromised host is especially vulnerable [
8].
Although widely studied as a safe vehicle for gene therapy, adeno-associated viruses (AAV) have infrequently been applied to treat cancers [
14]. Moreover, early work indicated human B-cells were poor targets for AAV [
9,
16,
26]. Consequently, little is known about AAV infection of human B-cell tumors, despite widespread derivation in recent years of AAV recombinants (rAAV) with altered tropisms [
5,
25].
Unlike oncolytic viruses, recombinant (r)AAV is nonpathogenic, does not independently replicate nor integrate into host cell DNA. The rAAV genome is eventually eliminated, reducing the risk of inadvertent spread to normal cells. Genes, including gene regulatory products engineered to eliminate only tumor cells, can be introduced into the AAV genome [
20]. Although an immune response to AAV-infected cells can impede gene therapy, this may prove advantageous in the setting of short-term anti-tumor therapy [
21], as often required for the spectrum of PTLD-associated tumors.
Herein, we examined and compared the in vitro ability of fifteen select recombinants derived from a well-characterized self-complementary (sc)rAAV plasmid vector that encodes enhanced green fluorescent protein (EGFP) [
16] to transduce human B-cells. The focus was on B-cells infected and immortalized with EBV. A rank order analysis of serotypes revealed rAAV6.2 transduction consistently supported EGFP expression in human B-cells and that the percentage of fluorescing cells was highest among B-cells bearing EBV. When herpes simplex virus type 1 (HSV1)-thymidine kinase (TK) was introduced into the rAAV6.2 genome, transduced human TK negative rhabdomyosarcoma cells were eliminated upon exposure to ganciclovir, and the viability of transduced B-cell tumor cells was reduced, providing support for development of rAAV-based oncolytic therapy. This proof-of-concept study demonstrates AAV serotypes such as rAAV6.2 preferentially transduce human EBV + B-cells and can support the expression of suicide genes, highlighting the potential for developing selective rAAV-mediated treatment of focal EBV + proliferative disorders.
Methods
Source of rAAV virions
Fifteen distinct serotypes of a single self-complementary rAAV-EGFP plasmid vector [
28] were produced by differential expression of capsid proteins at the Horae Gene Therapy Center of the University of Massachusetts Medical School. The parent rAAV-EGFP plasmid vector encodes a cytoplasmic enhanced green fluorescent protein (EGFP) driven by a hybrid CMV enhancer/chicken β-actin promoter [
27]. Following transduction, EGFP fluorescence can be directly visualized in cells by microscopy (confocal microscopy, flow cytometry) and protein expression can be detected with specific antibodies (see below). Each of the fifteen rAAV serotypes (Table
1) were synthesized in modified human HEK293 cells, extracted, purified, isolated, analyzed for genome content and titered as described [
25]. Production details for each of the fifteen serotypes are available through the Horae Gene Therapy Center.
Table 1
rAAV EGFP Serotypes
AAV1 | 1.8E + 13 |
AAV2 | 1.0E + 12 |
AAV3b | 6.0E + 12 |
AAV4 | 1.2E + 13 |
AAV5 | 1.4E + 13 |
AAV6 | 8.0E + 12 |
AAV6.2 | 8.0E + 12 |
AAV6TM | 8.0E + 12 |
AAV7 | 1.5E + 12 |
AAV8 | 7.0E + 12 |
AAV9 | 2.0E + 13 |
AAVrh8 | 8.0E + 12 |
AAVrh10 | 8.0E + 12 |
AAVrh39 | 1.0E + 13 |
AAVrh43 | 6.0E + 12 |
Source of cells
B-cell lines used in this study are listed in Table
2 together with their source. Primary B-cells were isolated from human spleen samples obtained from the New England Organ Bank in accordance with the policies of the Institutional Review Boards of the NEOB and of the University of Massachusetts Medical School. Primary B-cell isolation was performed by negative selection using the EasySep Direct Human B Cell Isolation Kit (STEMCELL Technologies) according to the manufacturer’s directions. Greater than 90% purity of the B-cell population was verified by flow cytometry using the monoclonal antibodies: APC-conjugated anti-CD20 (BioLegend) and FITC-conjugated anti-CD19 (BioLegend). All B-cells were maintained in RPMI-1640 (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal calf serum (HyClone), 100 U/ml penicillin, and 100 μg/ml streptomycin (Cellgro) at 37 °C in a 5% CO
2 incubator.
Table 2
Sources of Human B-cells
Primary cells |
Primary B-cell EBV- | New England Organ Bank | Human spleen |
B-LCL | | |
Newly EBV-infected primary B-cell | In vitro infection with B95-8 virus strain | Human spleen |
Standard B-lymphoblastoid cell line |
B95-8 | ATCC-CRL-1612 | Marmoset B-cells, immortalized with the prototype B95-8 EBV strain |
EBV + Burkitt lymphoma lines |
P3HR1 | ATCC-HTB-62 | Burkitt lymphoma derived from Jijoye Burkitt lymphoma |
Daudi | ATCC-CCL-213 | Burkitt lymphoma |
Raji | ATCC-CCL-86 | Burkitt lymphoma |
EBV-Burkitt lymphoma lines |
Ramos | ATCC CRL-1596 | Burkitt lymphoma |
BL41 | ATCC-ACC160 | Burkitt lymphoma |
In vitro-infected EBV + Burkitt’s lymphoma lines |
Ramos/B95-8 | Fred Wang and Elliot Kieff Harvard University | In vitro EBV-infected Burkitt lymphoma line |
BL41/B95-8 | Fred Wang and Elliot Kieff Harvard University | In vitro EBV-infected Burkitt lymphoma line |
Transduction procedures and confocal microscopy
To compare the transduction efficiency of each of the fifteen rAAV serotypes in each of the human B-cell lines, cells grown to mid-log phase were seeded in 24-well plates at a density of 2 × 105 cells per well. Twenty-four hours later, cells were transduced with an rAAV serotype at an MOI of 105. Forty-eight hours post-infection, fluorescence mediated by EGFP expression was captured by a ZEISS LSM 700 confocal microscope.
Transduction procedures and flow cytometry
B-cells from each of the described sources (Table
2) were grown to mid-log phase and then plated (3 × 10
5 cells/well) in 24-well plates. The cells were then transduced with each of the 15 rAAV serotypes at an MOI of 10
5 in independent experiments that were repeated in triplicate, n = 3. At 48 h post-infection, the cells were harvested, washed with PBS, fixed with 2% paraformaldehyde and analyzed by flow cytometry on an LSRII flow cytometer (BD Biosciences). The BD FACSDiva™ Software (BD Biosciences) was used to quantify the percentage of fluorescent (EGFP-expressing) cells.
Synthesis and production of rAAV6.2 encoding HSV1-TK
The rAAV-based plasmids encoding EGFP-HSV1-TK and HSV1-TK alone were synthesized by Gene Universal (Newark, Delaware). The respective genomes are displayed in Fig.
4a. The source of the inserts (EGFP-HSV1-TK and HSV1-TK) was previously described [
6]. Each genome was encapsidated to yield the rAAV6.2 serotype and produced for transduction experiments in the Horae Gene Therapy Center as described above.
The 143BTK- human rhabdomyosarcoma cell line, which lacks human TK-1 expression (ATCC), was pre-selected for bromodeoxyuridine resistance. Cells were maintained in DMEM (Corning) supplemented with 10% heat-inactivated fetal calf serum (HyClone), 100 U/ml penicillin, and 100 μg/ml streptomycin (Cellgro) at 37 °C in a 5% CO2 incubator. 143BTK cells were plated and transduced with either rAAV6.2-EGFP-HSV1-TK or rAAV6.2 HSV1-TK as described above for confocal analysis of B-cell lines.
Immunoblot confirmation of HSV1-TK protein expression in transduced cells
Selected B-cell lines grown to mid-log phase were seeded in 24-well plates at a density of 2 × 105 cells per well. Twenty-four hours later, cells were transduced with an rAAV6.2 serotype encoding HSV1-TK at an MOI of 105. Forty-eight hours after transduction, cells were harvested, lysed in RIPA buffer and quantified by Bradford assay. Total cellular protein (20 µg per lane) was separated by NuPAGE Bis–Tris (4–12%) polyacrylamide gel electrophoresis (Thermo Fisher Scientific) in MOPS buffer under reducing conditions. Protein was transferred onto a nitrocellulose membrane that was blocked with 5% skimmed milk in TBST for one hour prior to antibody incubation, five membrane washes and final detection with the ECL chemiluminescent detection kit, Clarity Max Western ECL Substrate (Bio-Rad).
The primary antibody rabbit polyclonal anti-HSV1-TK was generously provided by Dr. William Summers of Yale University. HRP-labeled goat anti-rabbit secondary antibody was used for final detection (Santa Cruz Biotechnology).
The primary antibodies, mouse anti-EGFP and mouse anti-GAPDH, were obtained from Santa Cruz Biotechnology, as were HRP-labeled goat anti-mouse IgG secondary antibodies used for chemiluminescent detection.
Sensitivity of HSV1‐TK‐transduced cells to GCV
Confocal images of 143BTK- cells transduced with rAAV6.2 HSV1-TK or with EGFP-HSV1-TK were collected using a ZEISS LSM 700 confocal microscope. Pictures were analyzed using the ZEN lite software. All pictures were taken at the same magnification (scale bar = 50 μm). Images were collected before and then 72 h after incubation with 10 μM ganciclovir. The concentration of ganciclovir displayed was established by a dose–response curve during prior experiments.
MTT viability analysis of rAAV6.2 HSV1-TK transduced B-cells incubated with ganciclovir
Selected B-cell lines (2 × 105 cells/ml) were cultured in 96 well microplates for 24 h at 37 °C. Cells were then transduced by rAAV6.2-HSV1-TK for 48 h. Transduced cells were incubated with 10 μM GCV or placebo (no GCV) for three days. After visualization by microscopy, twenty microliters of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (5 mg/ml) was added to each well and incubated with the cells for four hours. The accumulated formazan product was solubilized by adding 40 μl of DMSO. The optical absorption of the reaction product was measured at 570 nm. The percentage of surviving cells was calculated by measuring the mean absorbance of treated cells over the mean absorbance of untreated cells. Untreated cells were 100% viable. Results were represented as mean ± SD (n = 3).
Statistical analysis
Statistical analysis of fluorescence data generated by flow cytometry was performed with GraphPad Prism 8 software (GraphPad, San Diego, CA). Because there was minimal variation within conditions, we assumed all data followed a normal distribution. Values from independent experiments were reported as mean ± SD.
Ethical approval
This study was performed in accordance with the policies of the Institutional Review Boards of the NEOB and of the University of Massachusetts Medical School (IRB #H00004283).
Discussion
Among the most devastating consequences of successful hematologic and solid organ transplant is the development of PTLD. PTLD is a consequence of T-cell suppression required for engraftment of foreign cells and primarily results in uncontrolled outgrowth of EBV-bearing B-cells that run the gamut from normal appearing activated lymphocytes to aggressive clonal B-cell malignancies. Persons with other causes of T-cell deficiency (iatrogenic, AIDS, congenital diseases) are also at increased risk of these EBV-associated disorders. Rarely, PTLD can present as an EBV + T-cell outgrowth or an EBV + leiomyosarcoma. While aggressive surgery, chemotherapy, immunotherapy and radiation therapy variably control disease, therapy is often poorly tolerated by persons who are already profoundly immunosuppressed. PTLD may originate as a local growth—in Waldeyer’s ring, especially after a primary infection, in the B-cell associated infiltrate of an allograft, or as an isolated lymph node or a CNS lesion. Focused therapy that precisely targets individual lesions could significantly reduce treatment-based morbidity and mortality.
Among the severe manifestations of primary EBV infection is acute infectious mononucleosis (AIM) complicated by airway obstruction. This is caused by the exuberant proliferation of lymphocytes in the vicinity of Waldeyer’s ring. High-dose steroid therapy, while often effective, eliminates not only the EBV + B-cells but also other immune cells required to resolve the infection and thereby causes a clinical conundrum. rAAV administration raises the prospect of focused, effective therapy.
AAV as a therapeutic modality has been predominantly associated with gene replacement. In recent years, multiple innovations affecting the structure and content of the viral genome, the ability to alter and re-direct tropism by capsid mutation as well as utilization of new techniques that augment manufacturing [
25] has sparked interest in applying rAAVs to precision cancer therapy – systemic and targeted [
14]. Because initial studies indicated human B-cells were not susceptible to AAV infection, little published information was available relevant to cancers of B-cell origin. Nevertheless, current innovations suggested application of new rAAV variants to the treatment of B-cell tumors with emphasis on focal lesions amenable to direct introduction of comparatively small quantities of virus, would be valuable.
Results of the current proof-of-concept analysis revealed that among the fifteen rAAV serotypes evaluated, rAAV6.2 and those rAAVs closely related to 6.2 (rAAV6, rAAV6TM), based on capsid sequence, were most efficient in virus transduction. All fifteen rAAV candidates contained identical EGFP-encoding genomes, implicating differential encapsidation as the likely source of altered transduction efficiency. Although the percentage of cells transduced by rAAV6.2 varied between B-cell sources, transduction efficiency was highly reproducible in different assays and even when genome content was altered by the introduction of HSV1-TK. Primary B-cells were minimally transduced by all fifteen rAAV serotypes, though rAAV6.2 was most effective on a comparative basis. This result was entirely consistent with older observations that primary B-cells were not viable targets for AAV transduction. In contrast, B-cells that contained intact EBV genomes were among the most effectively transduced cells. Infection of primary B-cells and EBV negative cell lines by the prototype EBV B95-8 virus uniformly increased their susceptibility to rAAV6.2 transduction.
In addition to demonstrating the utility of rAAV6.2 as a therapeutic, these findings highlight the potential of this rAAV serotype to introduce nucleic acids into difficult to transfect human B-cell tumor lines to uncover oncogenic mechanisms. Optimization of transfection efficiency for each of the respective lines could be individually achieved.
The latent cycle EBV-encoded proteins, latent membrane proteins LMP-1 and LMP-2, mediate B-cell transformation upon B-cell activation of two main pathways, one that mimics T-cell stimulation of B-cells via CD40 and IL-4 (LMP-1), the other by mimicry of IgM receptor signaling (LMP-2) [
3,
10]. As a consequence, many B-cell surface antigens present at low levels on primary resting B-cells are upregulated or exposed, potentially increasing the access of rAAV6.2. While limited in number, studies by two groups lend weight to the hypothesis that a related activation event is key. Serial experiments conducted by the Hallek laboratory (2002–2004) using rAAV2 to transduce B-CLL cells showed that pre-incubating these cells with complexed CD40L, with anti-IgM (and to a lesser extent with CpG oligos) augmented rAAV2 transduction [
26]. Consistent with these observations, in 2018, Hung et al. showed that incubating primary human B-cells with a “B-cell activation cocktail” consisting of CD40L trimers, CpG and the interleukins-2, 10, 15 augmented rAAV6 transduction [
9]. Comparative analysis of eight rAAVs encoding EGFP (rAAV1, 2, 2.5, 5, 6, 8, 9, D–J), conducted by Hung et al. showed rAAV6 transduced 40% of cells, rAAV2 30%, and all others < 10%. Taken together, these disparate observations highlight a key role for CD40 signaling in regulating rAAV6.2-mediated B-cell transduction (though other activation signals may also contribute). The precise mechanism through which CD40 ligation augments subsequent transduction by these serotypes remains unknown. Modulation of a specific B-cell surface receptor (upregulation, altered access) or possibly an intracellular transit pathway is suggested on the basis of capsid specificity.
What does this mean for capsid specificity? Capsid sequence variation typically occurs on the external surface in hypervariable regions that comprise approximately 19% of the overall protein and determine tropism [
24]. Although tropism primarily reflects attachment to a cellular receptor(s), it can also comprise post-entry events that affect intracellular capsid stability, transport and nuclear delivery. While rAAV6.2 transduction was most efficient, the closely related serotypes rAAV6 and rAAV6TM capsids (clade A) were also effective. The AAV6.2 serotype was created by mutating the phenylalanine (F) residue at position 129 in the VP1 protein of AAV6 to leucine (L) and had also been found to increase transduction of other human cell types, such as human airway epithelium [
15]. To date, the precise mechanism by which transduction is enhanced is not known. The rAVV6TM (TM = triple mutant) capsid contains three mutations Y731F/Y705F/T492V that remove residues implicated in phosphorylation-dependent proteasomal degradation, an event with potential to impede nuclear delivery [
23]. However, the 6TM mutated capsid was not more efficient at human B-cell transduction. Similar to older studies, rAAV2 (clade B) ranked next in human B-cell transduction efficiency, though once again the mechanism remains unresolved.
The incorporation of HSV1-TK into the rAAV6.2 vector genome and successful demonstration of its activity post-transduction paves the way for exploration of additional suicide effectors as well as select EBV promoter/enhancer elements (e.g., EBV nuclear antigen-1, EBNA-1) that drive suicide effectors in a manner that guarantees expression only in targeted EBV + B-cell tumors [
13]. The observation that primary resting B-cells are not transduced is a plus. However, because rAAV6.2 has been shown to transduce other human cells, including certain cancers, a demonstration of selective tropism will be required for clinical translation. Further mutational analysis of the rAAV6.2 capsid to identify amino acid substitutions that confer selective tropism will, therefore, be necessary. Libraries of singleton mutations in the hypervariable region of the rAAV6 capsid are available for initial screening [
24], but multiple exchanges may be required. An approach that combines both vector and capsid alterations to create a highly selective rAAV6.2 variant has the potential to transform the treatment of localized EBV disease.
While development of a suitable in vivo model of focal EBV disease in rodents would confirm these results (despite caveats concerning EBV-rodent models) [
1], the observation that multiple murine cell types are susceptible to rAAV6.2 [
23] makes such an endeavor impracticable at this time. As discussed above, the development of a highly EBV-specific promoter able to activate a suicide gene or capsid mutations that produce exquisite cell specificity will be required.
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