(608) Anti-CD20 permits secondary lung gene transfer: implications for gene editing approaches in CF

Background: Approximately 8% of patients with CF, especially those not of European descent, have mutations unable to be rectified by current drugs. Inhaled gene therapy is one approach to target undruggable mutations. Gene therapy has several advantages over currently available drugs; most significantly, gene therapy can target any class of mutation. However prior of gene therapy trials have not been successful due to a combination of inefficient gene transfer, and simulation of immune responses which eliminate and prevent re-administration of the drug, causing insufficient and transient correction [1].

In the lung B cells play key roles as antigen presenting cells, particularly for larger non-soluble antigens such as viral particles [2], as well as precursors of plasma cells that are the source of neutralizing antibody. Prior work in the Kolls lab has shown that B cell depletion with αCD20 abrogates priming of lung memory T-cell responses to the fungus Pneumocystis murina [3]. We therefore hypothesized that administration of αCD20, a drug used to manage post-transplant lymphoproliferative disease in CF after lung transplantation, prior to gene transfer will block both humoral and cell-mediated immunity to viral vectors and allow for repeated vector delivery and increased transgene expression.

Methods: We selected an adenoviral vector (serotype 5) for gene delivery due to its well-known ability to elicit potent B cell and T cell immune responses. C57BL/6 mice were assigned to two experimental groups, which received either intraperitoneal αCD20 treatment or isotype control two days prior to the first round of Ad5-GFP vector delivery via intratracheal instillation. 4 weeks later, mice were dosed with Ad5-LacZ and sacrificed 3 days later to evaluate immune responses and second-round gene transfer efficiency. Systemic immune responses were assayed with IFNγ ELISPOT of splenocytes to recombinant Ad5 hexon protein. Finally, secondary gene transfer efficiency was determined by qRT-PCR for LacZ RNA in whole lung homogenate.

Results: After second round vector delivery, mice receiving αCD20 treatment had significantly higher levels of lung LacZ expression (Figure 1A) and significantly lower levels of Ad5-hexon responsive splenocytes (Figure 1B). Together, these data indicate αCD20 treatment attenuated the immune response against the adenoviral vector, allowing for higher levels of gene transfer.

Conclusions: αCD20 treatment prior to lung-directed vector delivery is a promising future direction for gene therapy, as temporary abrogation of immune responses may allow for repeated vector administration in a variety of contexts. Further research is needed to study dose optimization and duration of effects as well as test this regimen with other delivery systems including AAV vectors.

Acknowledgements: Supported by R35 HL139930 from NHLBI (JKK).

References: [1] G. U and A. EW, "Moving forward: cystic fibrosis gene therapy," Human molecular genetics, vol. 22, no. R1, 10/15/2013 2013, doi: 10.1093/hmg/ddt372.

[2] S. Hong et al., "B Cells Are the Dominant Antigen-Presenting Cells that Activate Naive CD4," (in eng), Immunity, vol. 49, no. 4, pp. 695-708.e4, 10 16 2018, doi: 10.1016/j.immuni.2018.08.012.

[3] W. Elsegeiny, T. Eddens, K. Chen, and J. K. Kolls, "Anti-CD20 antibody therapy and susceptibility to Pneumocystis pneumonia," Infection and immunity, vol. 83, no. 5, 2015 May 2015, doi: 10.1128/IAI.03099-14.