Lentiviral vectors (LVVs) are emerging as an enabling tool in gene and cell therapies, yet the toolkit for purifying them at scale is still immature. A pivoting moment in LVV isolation technology was marked by the introduction of affinity ligands for LVVs pseudo-typed with Vesicular Stomatitis Virus G (VSV-G) protein. Camelid antibody ligands were initially discovered and utilized to functionalize a resin with a capacity of 1014 LVV particles per liter (vp/L). Shortly thereafter, our team introduced VSV-G-targeting peptides and assessed their application as ligands for purifying LVVs from HEK293 cell harvests. In this study, we utilized these peptides to develop novel affinity resins and—first in this field—affinity membranes with optimal binding capacity, productivity, and removal of host cell contaminants. To that end, we evaluated resins of different material, particle and pore size, and functional density, as well as membranes with different fiber morphology, porosity, and ligand distribution. The lead peptide-functionalized resin and membrane featured high capacity (5 × 109 and 1.2 × 109 transducing LVV units per mL of adsorbent, TU/mL) and productivity (2.9 × 109 and 1.7 × 109 TU/mL min) and afforded a substantial enrichment of cell-transducing LVVs and reduction of contaminants (110–170-fold) in the eluates. Finally, we demonstrated an LVV purification process in four steps: clarification and nuclease treatment, affinity capture in bind-and-elute mode, polishing in flow-through mode, and ultra/dia-filtration and sterile filtration. The processes afforded yields of 33%–46%, a residual HCP level below 5 ng/mL, and productivity of 1.25–1.5 × 1014 active LVV particles per hour and liter of adsorbent.
There is a lack of effective lymphedema prevention methods. The objective of this study was to investigate the ability of decellularized lymph nodes (dLNs) transplantation to prevent hindlimb lymphedema.
Porcine dLNs were prepared using 1% sodium dodecyl sulfate and 1% Triton X-100, and the effectiveness of decellularization was assessed by histological assessment and DNA quantification. Lymph node (LN) fragments and dLNs were transplanted into mice, and samples were collected for evaluating biocompatibility at the fourth week postsurgery. Thirty-six SD rats were separated into a control group (lymphatic dissection), a dLNs group (lymphatic dissection and dLNs transplant) and a sham group (inguinal skin circumferentially incised). Hindlimb circumference was monitored every 3 days. Indocyanine green lymphography was performed before and every week after surgery. Samples were collected for histological assessment at the second and fourth weeks.
The dLNs showed virtually complete absence of cellular material, maintenance of spatial structures, and good biocompatibility and induced immune cell infiltration. Compared with that of the control group, the average hindlimb circumference of the dLN group was significantly reduced on postoperative days (PODs) 8, 12, and 16, and that of the sham group was significantly reduced on PODs 4, 8, 12, 16, and 20. The sham group exhibited intact inguinal LNs and lymphatic drainage. Neonatal lymphatic vessels (LVs) were observed in the dLN group, and obvious dermal backflow was observed in the control group. Transplanted dLNs induced the infiltration of immune cells, which subsequently integrated into the preexisting lymphatic system. Compared with those in the control group or sham group, the number of LYVE-1+ LVs in the affected limb was greater in the dLN group.
The dLNs scaffolds induced the infiltration of immune cells and promoted LVs regeneration, which integrated into the preexisting lymphatic system to accelerate the restoration of lymphatic drainage.
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