Background Despite vigorous and ongoing efforts, active immunizations have yet to induce broadly neutralizing antibodies (bNAbs) against HIV-1. An alternative approach is to achieve prophylaxis with long-term expression of potent biologic HIV-1 inhibitors with Adeno-associated Virus (AAV), which could however be limited by hosts' humoral and cellular responses. An approach that facilitates in vivo production of these complex molecules independent of viral-vectored delivery will be a major advantage. Methods We used synthetic DNA and electroporation (DNA/EP) to deliver an anti-HIV-1 immunoadhesin eCD4-Ig in vivo. In addition, we engineered a TPST2 enzyme variant (IgE-TPST2), characterized its intracellular trafficking patterns and determined its ability to post-translationally sulfate eCD4-Ig in vivo. Findings With a single round of DNA injection, a peak expression level of 80-100μg/mL was observed in mice 14 days post injection (d.p.i). The engineered IgE-TPST2 enzyme trafficked efficiently to the Trans-Golgi Network (TGN). Co-administrating low dose of plasmid IgE-TPST2 with plasmid eCD4-Ig enhanced the potency of eCD4-Ig by three-fold in the ex vivo neutralization assay against the global panel of HIV-1 pseudoviruses. Interpretation This work provides a proof-of-concept for delivering anti-HIV-1 immunoadhesins by advanced nucleic acid technology and modulating protein functions in vivo with targeted enzyme-mediated post-translational modifications. Funding This work is supported by NIH IPCAVD Grant U19 Al109646-04, Martin Delaney Collaboration for HIV Cure Research and W.W. Smith Charitable Trust.