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.