AAV-mediated Gene therapy offers great potential for curing Hemophilia B. Targets the liver cells, which are primarily responsible for producing FIX, the traditional gene therapy has its advantages. however, it still raises concerns. For example, a substantial injection of the virus may lead to an inconsistent distribution of the virus across different cell types and may cause harm to other vital organs and systems, including the brain, the circulation system etc. Although side-effects by using AAV vectors have been generally acceptable in humans, additional research is still required to evaluate the safety of the virus as it transduce to different tissue cells including liver cells. The ultimate determinant of the success of traditional gene therapy may be the dose-dependent genotoxicity to other organs and tissues. The higher the vector dose, the higher the success rate of gene therapy; however, it also increases the likelihood of liver genotoxicity [16] and distribution to other tissues and system such as brain, circulation system etc. [27].

In this study, we developed a novel AAV vector expressing hFIXco, designated DJ/8-hFIX, which has potential applications in the treatment of hemophilia B. Our results demonstrate that tr-HL7702 exhibits robust hFIXco secretion in vitro, which was consistent with Hashimoto H reported that GFP-tagged DJ/8-hFIX effectively expresses GFP when compared to other wild-type serotypes [28].

Based on the previously reported normal AAV vector baseline used in gene therapy [29], we arbitrarily selected a dosage of 1 × 1011 vg/kg of pure LP1-hFIX for intravenous injection into mice tail veins as positive control. Fortunately, all F9-KO mice that received this dosage survived, and their coagulation activities were observed to be significantly improved. When compared to wild-type mice, minimal significance was observed between the F9-KO mice and wild-type mice. For future studies, we plan to increase the LP1-hFIX dosage for pure gene therapy and prepare for clinical trials.

MSCs are fibroblastic and plastic-adherent cells that possess the ability to differentiate into various mesenchymal lineages, both in vitro [30, 31] and in vivo [32, 33]. Sung Jin Kim et al. observed that scAAV2 and scAAV5 were effective in transferring the GFP gene to human bone marrow-derived MSCs and umbilical cord blood-derived MSCs [34]. AAV-DJ/8 demonstrates efficient transduction across various cell types derived from diverse species and tissues, including primary human hepatocytes, melanoma cells, and embryonic stem cells [28]. Yunxia Zhang et al. employed a lentiviral vector to transduce IL-21 into HUCMSCs, which were then transplanted into SKOV3 ovarian cancer xenograft-bearing nude mice. This approach offers a promising novel therapeutic strategy for the clinical management of ovarian cancer [35]. Given the immunogenic properties and self-renewal potential of HUCMSCs, we propose that HUCMSCs could serve as an excellent host cell for AAV mediated gene therapy to delivering hFIXco. To date, there are no known reports that have demonstrated the use of HUCMSCs as host cell in AAV mediated gene therapy in Hemophilia B applications.

In this study, We successfully transducted the DJ/8-hFIX into HUCMSCs as a substitute for high-dose vector injection, and the tr-HUCMSCs secreting comparable stability and continuity of hFIXco in vitro and in vivo. Although the results are still preliminary, it holds significant promise for potential clinical applications due to its preliminary yet promising nature. The ultimate utility of this approach necessitates further development through rigorous and extensive pre-clinical studies prior to clinical trial verification.

The AAV-mediated cell-based gene therapy exhibits a significant advantage over traditional gene therapy approaches. In animal studies, the total DJ/8-hFIX dose for cell-based gene therapy protocol was 1 × 109 vg /kg. The total vector dosage used in this cell-based gene therapy was significantly lower than the traditional vector dosage (ranges from approximately 1 × 1011 to 1 × 1012 vg /kg). Lower dosages of vectors are employed in AAV-mediated cell-based gene therapy, simplifying the manufacturing processes and potentially leading to cost reduction in Hemophilia B treatment for future. Although this cell-based gene therapy has not yet been applied in humans, the significantly smaller total vector dosage used in this approach appears to offer greater safety compared to traditional gene therapy. By transducing the vectors into HUCMSCs in vitro without exposing them to other human tissue cells, our objective is to prevent inadvertent transduction of other tissue cells in vivo, thereby ensuring safety. HUCMSCs have been demonstrated to have minimal side effects in clinical trials. Our approach has the potential to avoid random AAV-mediated distribution to other tissues and system such as brain, circulation system etc. and also avoid liver genotoxicity induced by high-dose vector injection.

According to the report by Halder SK et al. [2], protein variants were most likely to be detrimental to protein structure and function, and further investigation is certainly needed into the structure of our purified hFIXco protein. However, studies in one Hemophilia B patient revealed that the activation of hFIXco secretion in tr-HUCMSCs is comparable with that of the positive control tr-HL7702. Although the observed increase in hFIX activity was relatively not very high which could be potentially attributed to the dilution of the cell supernatant, the elevation in FIX activity in this study is remarkable. Additionally, during this study, serum from a single Hemophilia B patient was obtained, and the results of coagulation time correction were provided an important evidence for its effectiveness. Despite the limited number of patients’ sera used, preliminary findings suggest that this study demonstrates, at least in part, that both tr-HUCMSCs and tr-HL7702 have the capacity to secrete hFIXco with coagulation correction potential.

Human MSCs were xenotransplanted into fetal sheep and integrated into different tissues including bone marrow, spleen, and liver [32]. Following a 2-week transplantation period, our experiments demonstrated the presence of HUCMSCs in bone marrow, spleen, and liver, particularly abundant in bone marrow, aligning with published reports [32]. This indicates that HUCMSCs maintain their functional properties, regardless of transduction status. Furthermore, tr-HUCMSCs may establish a foothold in the bone marrow microenvironment, exercising their secretory function for hFIXco. Following a five-month transplantation period, immunohistochemical analysis revealed the presence of weakly CD105-positive cells within the tissues. Hematoxylin-eosin staining confirmed similar tissue characteristics to the control group. This implies that the distribution of HUCMSCs or their transduced forms does not significantly impact tissue integrity, ruling out tumorigenic activity. The observed phenomenon may be attributed to the multilineage differentiation capabilities of HUCMSCs, facilitating their integration into the surrounding tissue.

Studies on cutting tail experiments of F9-KO mice revealed that the transgene hFIXco was effective in both direct injection and tr-HUCMSC treatment. The therapeutic effect of the transgene is achieved by minimizing bleeding episodes. Mazumder TH et al. [36] reported that evolutionary forces and genetic relationships can influence the codon utilization bias of a gene. The Calcium-binding EGF-like domain (residues 93–125), which may be concerned as crucial for FIX function, exhibits a high degree of similarity between hFIX and mFIX protein, differing by only 5 amino acids (supplementary Material 6). This may explain the therapeutic effect by shortened bleeding for F9-KO mice.

Giang N. Nguyen et al. [15] revealed AAV gene therapy in dogs with hemophilia A identifies several clonal expansions of transduced liver cells after 10 years observation. Our Hematoxylin-eosin staining results in NSG mice, however, did not show any clonal expansions of transduced cells in tissues including liver, spleen, lung, and brain after 5 months of experimentation. Although we did not investigate the genomic integrations in this study, the experiment may still provide some evidence for the safety of DJ/8-hFIX/ tr-HUCMSCs-mediated gene therapy.

A notable disparity was observed in the activity of hFIX detected in NSG mice that received injections of DJ/8-hFIX or 0.9% NS. This finding suggests that the detection of hFIX activity system, which was conducted in the human factor IX deficient plasma by employing a phospholipid (origin from rabbit brain powder) surface activator under the working concentration (0.025 mol/L) of CaCl2 is relatively specific to human FIX and is not much sensitive to mouse FIX. The results of human FIX activity in the sera of F9-KO mice 60 days post-injection were consistent with the findings obtained from NSG mice.

It is intriguing to observe that the F9-KO mice maintained excellent health throughout the experiment despite receiving tr-HUCMSCs without eliciting an immune response. The mechanism underlying the absence of an immune response against HUCMSCs in F9-KO mice remains enigmatic. One plausible explanation is the extremely low immunogenicity of HUCMSCs. Another potential factor could be the inherent characteristics of the F9-KO mice strain (C57BL/6JSmoc-F9em1Smoc). Report [37] cites the discovery of a mutation in the commercially available C57BL/6 strain, specifically gene duplication, that impairs the Dock2 gene and subsequently results in immune deficiency. The underlying mechanism of this phenomenon merits further investigation.

We have successfully transduced the HUCMSCs with the novel DJ/8-hFIX vector. Furthermore, a stable and consistent secretion of hFIXco was observed in the transduced HUCMSCs. The biological activity of secreted hFIXco was assessed both in vivo and in vitro. Notably, our study did not identify any significant differences between the direct injection of the DJ/8-hFIX vector and the one mediated via the HUCMSCs. Animal models have demonstrated the safety of this cell-based gene therapy, paving the way for its clinical application. While the disease-free survival observed at five months post-injection suggests the safety of HUCMSC-based DJ/8-hFIX gene therapy, and histological staining with hematoxylin-eosin reveals normal tissue morphology, the integration of the DJ/8-hFIX gene into host genomic DNA remains unclear. Additional research is required to investigate the presence of anti-DJ/8-hFIX antibodies in patients during clinical trials. The precise mechanism underlying the internalization of the DJ/8-hFIX vector by HUCMSCs and the secretion of active hFIXco remains enigmatic. Extensive research is necessary to elucidate this issue.