Feline leukemia virus subgroup B uses phosphate transporters and shows promise as an envelope-based gammaretroviral vector for gene therapy in dogs

Feline leukemia virus subgroup B uses phosphate transporters and shows promise as an envelope-based gammaretroviral vector for gene therapy in dogs | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Feline leukemia virus subgroup B uses phosphate transporters and shows promise as an envelope-based gammaretroviral vector for gene therapy in dogs Didik Pramono, Karin Inoue, Miharu Nishimoto, Loai AbuEed, Takuya Mizuno, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8217702/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Chimeric antigen receptor (CAR)-T cell therapy, a cellular immunotherapy, has attracted considerable attention as a cancer treatment modality. Several CAR-T cell therapies have been approved for human use; however, this technology has limited application in veterinary medicine. Cancer is a notable health concern in dogs, with over 50% of dogs aged > 10 years developing the disease. Viral vectors are useful for gene transfer and cellular protein expression, have high transfer efficiency, and can be expressed sustainably in the host. Particularly, retroviral vectors have received much attention because their genome can be introduced into the host genome, making them useful for delivering therapeutic genes into the host body. In this study, we investigated the efficiency of feline leukemia virus subgroup B (FeLV-B) Env-pseudotyped virus vectors for gene transfer compared with GaLV, KoRV, HPG, and WMV Env-pseudotyped viruses. Canine cells and cells expressing canine phosphate transporters (canPit1 or canPit2, entry receptors of FeLV-B) were infected with Env-pseudotyped viruses, and the infectious titer was measured. Additionally, cells that were persistently infected with retroviruses were identified, and receptor interference experiments were performed to investigate FeLV-B receptor utilization. Our findings revealed that FeLV-B/ON-T and FeLV-B/B16 Env-pseudotyped viruses exhibited notably higher infectious titers than other viruses. However, this study was limited to in vitro infections using cell-based assays. Further investigations are required to determine the efficiency and safety of in vivo gene transfers. Overall, this study highlights retroviral tropism in dogs and provides information on gene therapy systems. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Gene therapy modifies genes by adding new genes, editing existing genes, or silencing problematic genes. There are two types of gene therapy: in vivo gene therapy, in which a vector containing a gene is directly administered to the patient, and ex vivo gene therapy, in which a gene is introduced into cells taken from the patient and subsequently administered [ 1 ]. Research efforts are underway on various diseases, including cancer, viral infections, and genetic diseases [ 2 ]. Immunotherapy options used in humans have not yet been translated into veterinary medicine. A representative example is chimeric antigen receptor (CAR)-T cell therapy, which has recently shown good results in humans against hematopoietic malignant tumors and has been approved as a treatment for various B-cell malignant tumors and multiple myeloma [ 3 ]. However, these advancements have limited benefits for canines [ 4 ].A preliminary study investigated the use of amphotropic murine leukemia virus (4070A MuLV) and gibbon ape leukemia virus (GALV) in canine tumor models [ 5 ]. Retroviral and lentiviral vectors are the primary gene transfer technologies used in CAR-T cell therapy. The introduced gene is maintained on the chromosome, even after cell division, via insertion into the chromosome. Replication-incompetent viruses are used for gene transfer and genome integration [ 6 ]. In this study, we focused on developing viral vectors using Feline Leukemia Virus subgroup B (FeLV-B). Considerable basic research has been conducted on FeLV-B infection. Viral antigens, antibodies against the virus, and viral genes can be detected using enzyme-linked immunosorbent assay and polymerase chain reaction (PCR), which have high sensitivity and specificity. A vaccine has also been developed against FeLV-B. FeLV-B is a promising and safe viral vector candidate for clinical studies [ 7 , 8 ]. FeLV is a gammaretrovirus commonly found in domestic cats worldwide [ 9 ]. FeLV is associated with a high mortality rate in domestic cats and has been reported to induce malignant hematopoietic disorders, including lymphoma, myelodysplastic syndrome, acute myeloid leukemia, aplastic anemia, and immunodeficiency [ 10 , 11 ]. FeLV subgroup A (FeLV-A) is the primary horizontally transmitted virus in domestic cats [ 12 ]. FeLV-B results from recombination in the env region between FeLV-A and endogenous FeLV present in the feline genome [ 12 ]. FeLV-B uses phosphate transporters 1 (Pit1) and 2 (Pit2) as entry receptors [ 13 – 15 ]. In addition to FeLV-B, various other viruses, such as koala retrovirus (KoRV; koalas), Gibbon ape leukemia virus (GaLV; Gibbons), Hervey pteropid gammaretrovirus (HPG; bats), 4070A amphotropic murine leukemia virus (4070A MuLV; murine), 10A1 MuLV (murine), and woolly monkey virus (WMV; monkeys), use Pits as viral receptors and can spread to hosts. Interspecies transmission may also occur [ 15 ]. Only a few cases of retroviral infections have been reported in dogs. In vivo infection in dogs showed the same result: no infection by the feline endogenous retrovirus RD-114 [ 16 ]. Notably, cultured dog cells are susceptible to retroviral infections, such as RD-114 and 4070A MuLV [ 17 , 18 ]. Although FeLV-B infects canine cell lines [ 14 ], there have been no reports of FeLV-B receptors in dogs. In this study, we investigated the efficiency of FeLV-B Env-pseudotyped virus vectors for gene transfer in canine cells compared with GaLV, KoRV, HPG, and WMV Env-pseudotyped viruses. To ensure the safety of gene transfer methods using retroviral vector systems, it is necessary to identify retroviral receptors. Materials and methods Isolation of canine Pit (canPit)1 and 2 and construction of the expression vector RNA was isolated from canine peripheral blood mononuclear cells (PBMCs) and canine mammary tumors (KwDM) [ 15 ] using the RNeasy kit (Qiagen, Hilden, Germany), and the extracted RNA was treated with recombinant DNase I (RNase-free; Takara, Shiga, Japan). Complementary DNA (cDNA) was synthesized using the PrimeScript II First-Strand cDNA Synthesis Kit (TaKaRa, Shiga, Japan) with oligo(dT) primers. canPit1 cDNA was amplified (based on the predicted sequence; XM_038691442.1) by PCR using the first primer pair Fe-923S and Fe-943R. canPit2 cDNA was amplified (based on the predicted sequence; XM_038689720.1) by PCR using the first primer pair, Fe-924S and Fe-944R (Table S1 ). PCR products were digested with BamH1 and Xho1 for canPit1 and EcoR1 and BglII for canPit2, and each fragment was inserted into the pMSCVneo retroviral vector (TaKaRa). KOD-ONE Blue polymerase (Toyobo, Osaka, Japan) was used for PCRs according to the manufacturer’s instructions. The resulting plasmids were confirmed by sequencing (Fasmac Corporation, Atsugi, Japan). Cell lines In this study, we used the following cell lines: canine hepatocellular carcinoma (HCC) cells, canine mammary (KwDM) cells [ 19 ], Mus dunni tail fibroblast (MDTF) cells [ 20 ], GPLac (an env-negative packaging cell line containing the MuLV gag-pol gene and beta-galactosidase [LacZ]-coding pMXs retroviral vector) [ 19 ], MDTF cells expressing canPit1 (MDTF-canPit1), and MDTF cells expressing canPit2 (MDTF-canPit2). Cells were cultured in Dulbecco’s modified Eagle’s medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal calf serum and 1X penicillin–streptomycin and subsequently incubated in a CO 2 incubator at 37°C. Detection of canPit1 and canPit2 expression levels via RT-qPCR RNAs from canine PBMC, KwDM, and HCC cell lines were extracted as described in the section “Isolation of canPit1 and canPit2” above. Briefly, cDNA was amplified using SYBR Premix Ex Taq II (Tli RNase H Plus; Takara) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). canPit1 was amplified using primers DA333046-F and DA333046-R. canPit2 was amplified using primers DA147288-F and DA147288-R and the internal control canine glyceraldehyde-3-phosphate dehydrogenase was amplified using the primers Fe-1011S and Fe-1025R as previously described [ 21 ]. Thermal cycling was performed according to the manufacturer’s instructions. Establishment of cell line expressing canPit1 and canPit2 PLAT-A (amphotropic MuLV) packaging cells were transfected with expression vectors (pMSCVneo-canPit1, pMSCVneo-canPit2, pMSCVneo-huPit1, or pMSCVneo empty vector) using TransIT®-293 reagent (Takara). PLAT-GP packaging cells were co-transfected with MuLV 10A1 and pMSCVneo-huPit2 to establish the huPit2 cell line. After 2 days, the supernatants were collected, filtered through a 0.22 µm filter, and used to inoculate MDTF in the presence of polybrene (10 µg/mL; Nakarai). The cells were cultured in a medium containing 600 µg/mL neomycin (G418) for 2 weeks. These cells were termed MDTF-canPit1, MDTF-canPit2, and MDTF-empty. Isolation of Env FeLV-B16 Briefly, FeLV-B16 was isolated from 293T/ON-T #3 cells [ 22 ] using specific primers Fe-227S and Fe-919R. Thereafter, the amplicon was cloned into the TOPO vector. The plasmid was sequenced by Fasmac Corporation (Atsugi, Japan). Env was amplified using Fe-944S and Fe-958R based on the FeLV-B/B16 proviruses in the TOPO vector and cloned into pFUΔss between the Bam HI and Eco RI restriction sites. Env expression vectors were constructed using KOD-ONE Blue (Toyobo, Osaka, Japan) according to the manufacturer’s protocol. The resulting Env expression plasmids were confirmed by sequencing (Fasmac Corporation). Env-pseudotyped virus preparation Env-pseudotyped viruses carrying LacZ as a marker were prepared as previously described [ 19 ]. Briefly, GPLac cells were seeded in a six-well plate at a concentration of approximately 1 × 10 6 cells/well 1 day prior to transfection. Notably, the Env expression plasmids used for viral preparation were pFUΔss GaLV Env, pFUΔss KoRV Env (opt-KoRV-A2), pFUΔss HPG Env (opt-HPG), pFUΔss WMV V655 Env [ 15 ], pFUΔss FeLV-B/GA, pFUΔss FeLV-B/ON-T, pFUΔss FeLV-B/MZ40-5B, pFUΔss FeLV-B/KG20-5B, pFUΔss FeLV-B/FO36-5B [ 23 ], pFUΔss FeLV-A/clone33 [ 24 ], FeLV-B/B16, opt-KoRV-A2 Env, opt-HPG, opt-WMV V655 [ 15 ], MuLV 10A1, and 4070A Env (Novus Biologicals). At 48 h after transfection using TransIT-293 transfection reagent, the culture supernatants were collected, filtered through a 0.22-µm filter or centrifuged at 15,400 ×g for 2 min at 4°C, and stored at − 80°C as virus stock for further experiments. Viral titer of Env-pseudotyped virus Viral supernatants were collected from virus-producing cell lines (see the Env-pseudotyped virus preparation section) and centrifuged to remove cells and debris. Genomic viral RNA was purified from a small aliquot of the supernatant using the NucleoSpin RNA Virus Kit (Takara). RNA was then treated with DNase I to remove any residual plasmid DNA that may have been carried over from the transient transfection of packaging cells. Viral RNA samples were subjected to quantitative reverse transcription PCR (qRT-PCR) to determine the viral titer using Retro-XTM qRT-PCR (Takara) following the manufacturer’s instructions. Viral infection assay Target cells were seeded in 24-well plates at a concentration of 1–3 × 10 4 cells/well 1 day prior to infection. Thereafter, the cells were infected with 250 µL of Env-pseudotyped virus in the presence of 10 µg/mL of polybrene (Nacalai Tesque, Kyoto, Japan) for 2 h. After adding fresh medium, the cells were cultured for 2 days post-infection. After incubation for 48 h, the supernatants were removed, and the cells were fixed with 250 µL of 2% glutaraldehyde for 15 min at room temperature (20–25°C). Thereafter, the cells were stained with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, and blue-stained nuclei were counted under a microscope. Viral titers are presented as infectious units (IU) per mL, with standard deviations. MDTF cells expressing only the vector were used as negative controls. Virus recombination analysis Briefly, recombination events were assessed using RNA-seq data obtained from HCC and KwDM cell lines infected with FeLV-B. Reads were trimmed using Trimmomatic (v0.39) [ 25 ] to remove adaptors and low-quality sequences. Thereafter, the trimmed reads were aligned to a combined genome of Canis lupus (GCA_011100685.1) and FeLV-B (LC861716) using HISAT2 (v2.2.1) [ 26 ]. Alignments were coordinate-sorted and indexed using SAMtools (v1.22.1) [ 27 ]. Phylogenetic and sequencing analysis A phylogenetic tree was constructed using the sequences listed in the Accession Numbers section. MUSCLE was used to align amino acid sequences for phylogenetic analysis [ 28 ], and all other procedures were performed using the MEGA11 software package [ 29 ]. For the phylogenetic analysis of Pit1, Pit2, and related proteins of gammaretrovirus receptors, the neighbor-joining method [ 30 ] was employed to construct a phylogenetic tree with amino acid substitutions in the JTT model [ 31 ]. The robustness of the trees was evaluated by bootstrapping 1,000 times [ 29 ]. Results Isolation and characterization of canPit1 and canPit2 In this study, we investigated canPit1 and canPit2 as mammalian retroviral receptors. Pit1 and Pit2 are members of the solute carrier protein families SLC20A1 and SLC20A2, respectively. Initially, we isolated canine cDNA from canine KwDM cells and PBMCs using qRT-PCR, which were referred to as the predicted canPit1 (XM_038691442.1) and canPit2 (XM_038689720.1), respectively. Molecular cloning of PCR-positive samples was performed to obtain the canPit1 and canPit2 open reading frames. Our isolates and predictions for canPit1 and canPit2 in the database were 100% identical. Alignments of the amino acid sequences of the proteins encoded by the isolated canPit1 and canPit2 sequences are shown in Figs. 1 and 2 . canPit1 and canPit2 were predicted to encode proteins with 687 and 653 amino acids, respectively, with approximately 89.2% similarity. Additionally, we conducted a phylogenetic analysis comparing our canPit1 and canPit2 clones to related sequences of Pit1 and Pit2 in other species and found that canPit1 and canPit2 receptors were classified in the same group (Fig. 3 A). Expression of canPit1 and canPit2 in canine tissues and cell lines In the present study, we evaluated the expression of canPit1 and canPit2 in KwDM cells, HCC cells, and PBMCs. Notably, all samples exhibited canPit1 and canPit2 expression (Fig. 3 B). Although canPit2 was barely expressed in KwDM cells, CanPit1 and canPit2 were highly expressed in HCC cells. Additionally, canPit1 and canPit2 showed similar expression levels in PBMCs. Overall, these findings indicate that canPit1 and canPit2 are widely expressed in canine tissues and cell lines. Evaluation of canPit1 and canPit2 in retroviral infection In this study, we conducted infection assays by infecting canine cell lines (KwDM and HCC) with FeLV-B Env-pseudotyped viruses and non-feline mammalian retroviruses (GaLV, KoRV-A, HPG, and WMV). To ensure that we used the same viral titer, we assessed the titer of the Env-pseudotyped virus (Fig. S1 ). As expected, all Env-pseudotyped viruses could infect KwDM and HCC cell lines with FeLV-B/B16 and exhibited high viral titers (Fig. 4 A). Additionally, we investigated whether the infection required canPit1 or canPit2. MDTF cells were used because they are resistant to viral infections. MDTF-canPit1 and MDTF-canPit2 cells were constructed and used as target cells. Importantly, MDTF-canPit1 cells were permissive to all Env-pseudotyped viruses tested. The viral titers for Env-pseudotyped viruses varied (1,000–100,000 IU/mL), with FeLV-B Env-pseudotyped viruses exhibiting high viral titers and WMV V66 showing the lowest viral titers (Fig. 4 B). In contrast, MDTF-canPit2 was only permissive to FeLV-B/(ON-T and B16), GaLV, KoRV-A, and HPG, but not to FeLV-B (MZ40-5B, KG20-5B, and FO36-5B) and WMV V655. Notably, GaLV showed highly permissive infectivity, with viral titers reaching 10,000 IU/mL. MDTF cells expressing only the vector (negative control) were not infected with Env-pseudotyped viruses. FeLV-B/ (GA, MZ40-5B, and KG20-5B) and WMV V655 could only use canPit1, whereas FeLV-B (ON-T and B16), GaLV, KoRV-A, and HPG could use both canPit1 and canPit2 for infection (Fig. 4 B). Overall, our results suggest that FeLV-B (ON-T and B16) has the highest infectivity for canPit1 and canPit2. In addition, the pseudotyped FeLV-B/B16 did not infect KwDM or HCC cells infected with FeLV-B/GA. Conversely, FeLV-B/GA did not infect KwDM or HCC cells infected with FeLV-B/B16 (Fig. 4 C and D). Collectively, these results suggest that blocking the canPit1-mediated infection pathway prevents FeLV-B/B16 infections, highlighting the role of the pathway in FeLV-B/B16 infection. FeLV-B/B16 recombination analysis To investigate whether FeLV-B/B16 can recombine with the canine genome, RNA-seq reads from HCC and KwDM cell lines infected with FeLV-B/B16 provirus were aligned to a combined C . lupus and FeLV-B genome. Notably, the overall alignment rates were 95.86% (HCC) and 94.91% (KwDM). Additionally, the FeLV-B genome showed strong transcription, with 627,648 and 918,306 mapped reads in HCC and KwDM, respectively. However, split-read analysis from SAM supplementary alignments revealed no virus–host (FeLV↔dog) junctions and no intra-viral (FeLV↔FeLV) rearrangements within the detection thresholds (≥ 3 unique split reads, mapping quality scores (MAPQ) ≥ 20, breakpoint concordance within ± 5 bp) (32–34), indicating that recombination between FeLV-B and the canine genome did not occur. Discussion In humans, immunotherapy is widely used to treat various diseases, most notably cancer and autoimmune diseases. Developments such as tumor-specific CAR-T cells have broadened treatment options for human diseases, including blood cancers, specifically certain lymphomas, leukemias, and multiple myeloma [ 35 , 36 ]. However, animals, particularly canines, have not benefited from these advancements. The retroviral system is the main method used in gene transfer technologies for CAR-T cell therapy, similar to the lentiviral system. In this study, we propose an alternative retroviral system for delivering gene targets using feline retrovirus (FeLV-B). FeLV-B has been extensively characterized and investigated for its role in the pathogenesis of viral infections, implying that it is a promising and safe viral vector for use in therapy. Many retroviral envelope proteins bind to membrane transporters to enter the host for infection, including THTR1, which is used by KoRV-B [ 37 ] and FeLV-A [ 38 ]. Pit1 has been described as an entry receptor for both GaLV and KoRVA [ 39 , 40 ]. This has sparked research interest in their potential to enable viral entry into immune cells. The expression patterns of various receptors on host cells enable viruses to infect cells at specific stages of activation and proliferation [ 41 ]. Additionally, we investigated FeLV-B infection and found a high titer in canine cells, with canPit1 and canPit2 as entry receptors for infection. FeLV-B/ON-T and FeLV-B/B16 have higher titers of GaLV infection, which is commonly used for gene therapy, than other gammaretroviruses [ 42 , 43 ]. CAR-T therapy products, such as axicabtagene ciloleucel (Yescarta, Kite Pharma Inc.; FOSUN Kite) and brexucabtagene autoleucel (Tecartus, Kite Pharma, Inc.), use a gamma retroviral vector (a murine stem cell virus-based vector pseudotyped with a GaLV envelope) [ 44 ]. KoRV-A pseudotyped lentiviral vectors have also been investigated for gene transfer into isolated immune cells [ 41 ]. Our findings revealed that FeLV-B/ON-T and FeLV-B/B16 had higher titers than KoRV-A and GaLV. To corroborate this finding, we evaluated the expression of canPit1 and canPit2 and found high expression levels in two cell lines (KwDM and HCC) and canine PBMCs. These results support the notion that FeLV-B uses canPit1 and canPit2 as entry receptors for infection. Moreover, no recombination occurred between FeLV-B/N16 and the canine genome, as demonstrated by the infected replication-competent virus in HCC cells. Collectively, our findings indicate that the gammaretroviral system is a promising alternative to gene or CAR-T cell therapy. FeLV-B, particularly FeLV-B/ON-T and FeLV-B/B16, are promising candidates. In conclusion, we propose the use of FeLV-B Env as a viral vector system for gene therapy and demonstrate that FeLV-B Env-pseudotyped viruses can efficiently infect cells via Pit1 and Pit2. However, to investigate the cell introduction efficiency in more detail, their infectivity in newly isolated tumor cells and lymphocytes must be examined. Additionally, gene introduction using retroviral vectors carries the risk of carcinogenesis due to gene insertion into chromosomes, introduction into germ cells, and acquisition of proliferation ability in viral vectors [ 45 ]. Moreover, in vivo safety could not be established in the present study. However, extensive basic research has been conducted on FeLV-B. Given that viral antigens and antibodies can be detected and a vaccine for FeLV-B has been developed [ 46 , 47 ], FeLV-B is expected to be a safe viral vector for therapy. However, the efficiency and safety of introducing viral vectors during in vivo gene therapy in dogs and other animals must be examined in the future. Declarations Competing interests The authors have no relevant financial or non-financial interests to disclose. Ethical approval Animal studies were conducted in accordance with the guidelines for the care and use of laboratory animals provided by the Ministry of Education, Culture, Sports, Science, and Technology, Japan. All the experiments were approved by the Genetic Modification Safety Committee of Yamaguchi University. Funding This study was funded by the Japan Society for the Promotion of Science KAKENHI (Grant numbers: 23H02393 and 23K27086). KN received the funding. The funders had no role in the study design, data collection and interpretation, or the decision to submit the manuscript for publication. Author contributions Kazuo Nishigaki conceptualized the study. Data curation was performed by Didik Pramono, Karin Inoue, Miharu Nishimoto, and Kazuo Nishigaki. Formal analysis of collected data was performed by Didik Pramono, Karin Inoue, and Kazuo Nishigaki. The funding for the study was acquired by Kazuo Nishigaki. The research/investigation was performed by Didik Pramono, Karin Inoue, Miharu Nishimoto, Loai AbuEed, and Kazuo Nishigaki. Research methodology was designed and implemented by Didik Pramono, Karin Inoue, Miharu Nishimoto, Loai AbuEed, Takuya Mizuno, Ariko Miyake, and Kazuo Nishigaki. Project administration and supervision were performed by Kazuo Nishigaki. Research validation was performed by Didik Pramono, Karin Inoue, Miharu Nishimoto, Ariko Miyake, and Kazuo Nishigaki. The original draft of the manuscript was written by Didik Pramono and Kazuo Nishigaki. Acknowledgments We are grateful to Dr. Masaharu Hisasue for HCC cells, Dr. Yoshinao Kubo for MDTF cells, and Dr. Toshio Kitamura for GP cells and the pMxs retroviral vector. This study was funded by the Japan Society for the Promotion of Science KAKENHI (Grant numbers: 20H03152 and 23K27086). KN received the funding. The funders had no role in the study design, data collection and interpretation, or the decision to submit the manuscript for publication. Data availability statement The accession numbers for publicly available data used in this study are as follows: Felis catus ASCT1 (NM_001278844), Felis catus ASCT2 (XM_045045413), gibbon Pit1 (XM_032176718), rat Pit1 (NM_031148), mouse Pit1 (NM_015747), human Pit1 (NM_005415), gibbon Pit2 (XM_032143780), rat Pit2 (NM_017223), and human Pit2 (NM_001257180). 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09:57:30","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":122947,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8217702/v1/a91350b111ea39317b757ed6.html"},{"id":97429659,"identity":"7cccf05e-3901-41b0-a9ad-c76598d11621","added_by":"auto","created_at":"2025-12-04 09:57:30","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":810859,"visible":true,"origin":"","legend":"\u003cp\u003eAmino acid sequence alignment of canine, human, gibbon, rat, feline, and mouse Pit1.\u003cstrong\u003e \u003c/strong\u003eConserved amino acid residues are indicated by dots and positions with differences are shown as letters. Gaps in the amino acid sequence are indicated by hyphens. The putative transmembrane domains (boxes) were predicted based on human Pit1 sequence. Pit1, phosphate transporter 1\u003c/p\u003e","description":"","filename":"Figure1caninepaper.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8217702/v1/4cbd9f8071d36e0728e48474.jpg"},{"id":97429665,"identity":"f7ca5cbe-9490-490c-970d-01fadf7ccb19","added_by":"auto","created_at":"2025-12-04 09:57:30","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":807872,"visible":true,"origin":"","legend":"\u003cp\u003eAmino acid sequence alignment of canine, human, gibbon, rat, feline, and mouse Pit1. Conserved amino acid residues are indicated with dots, while positions with differences are shown as letters. Gaps in the amino acid sequence are indicated with hyphens. The putative transmembrane domains (boxes) were predicted based on human Pit2. Pit2, phosphate transporter 2\u003c/p\u003e","description":"","filename":"Figure2caninepaper.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8217702/v1/b8a1215436879d919340a90f.jpg"},{"id":97429657,"identity":"6a991762-e2c0-4901-8905-09716b36a7c9","added_by":"auto","created_at":"2025-12-04 09:57:30","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":307518,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis.(A) Phylogenetic tree of canine Pit1, Pit2, and related proteins with gammaretrovirus receptors.\u003cstrong\u003e \u003c/strong\u003eThe amino acid sequences of receptors were used to construct a neighbor-joining tree. The scale bar indicates the evolutionary distance in amino acid substitutions per site. The scale bar indicates the evolutionary distance in substitutions per site. The samples in this study are indicated in red. (B) Canine Pit1 and Pit2 expression levels. Quantification of transcripts using quantitative RT-PCR in canine PBMC. The x-axis indicates the analyzed samples. The y-axis indicates the expression level normalized to that of GAPDH. Normalized expression in PBMCs is shown as 1. Pit, phosphate transporter; RT-PCR, Reverse transcription polymerase chain reaction; PBMC, Peripheral Blood Mononuclear Cell; GAPDH, Glyceraldehyde 3-Phosphate Dehydrogenase; KwDM, canine mammary tumor; HCC, Hepatocellular Carcinoma\u003c/p\u003e","description":"","filename":"Figure3caninepaper.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8217702/v1/0cff118a6eb80c8b09a1752e.jpg"},{"id":97666849,"identity":"5b2972c7-3fec-49e0-8834-4987e1ee2958","added_by":"auto","created_at":"2025-12-08 09:22:13","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":479147,"visible":true,"origin":"","legend":"\u003cp\u003eInfectivity and receptor usage of FeLV-Bs in canine cells. (A) Infectivity of FeLV-B (GA, MZ40-5B, KG20-5B, FO36-5B, ON-T, and B16), GALV, KoRV-A, HPG, WMV V655 Env-pseudotyped viruses. (B) Infection assay of FeLV-B (GA, MZ40-5B, KG20-5B, FO36-5B, ON-T, and B16), GALV, KoRV-A, HPG, and WMV V655 Env-pseudotyped viruses in MDTF-canPit1, MDTF-canPit2, and MDTF (empty vector) as target cells for receptor usage. (C and D) Interference assay of FeLV-B/GA and FeLV-B/B16. KwDM (C) or HCC (D) cells, pre-infected with FeLV-B/GA, or FeLV-B/B16, were infected with the Env-pseudotyped viruses. Viral titers are indicated on the x-axis of the graph. Infectious units were determined by counting the number of log10-galactosidase (LacZ)-positive cells per milliliter of virus, as indicated on the y-axis. Virus infection titers with standard deviations were averaged from three independent experiments. The results of the control in this study (C and D) are derived from Figure 4A. FeLV-B, Feline Leukemia Virus subgroup B; GALV, Gibbon Ape Leukemia Virus; KoRV-A, Koala Retrovirus A; HPG, Hervey pteropid gammaretrovirus; WMV, Woolly Monkey Virus; MDTF, Mus Dunni Tail Fibroblast; Pit, Phosphate Transporter; KwDM, canine mammary tumor; HCC, Hepatocellular Carcinoma\u003c/p\u003e","description":"","filename":"Figure4caninepaper.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8217702/v1/d3776c6476cf5df6be3f66c8.jpg"},{"id":97677643,"identity":"f12e625d-f760-444c-a2e0-2d4bec40c97e","added_by":"auto","created_at":"2025-12-08 09:53:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3148685,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8217702/v1/cc60eee6-986c-47a8-a99d-282cfd1f5a18.pdf"},{"id":97429661,"identity":"d2ef2401-17b5-42e6-b07a-485d5160ea7d","added_by":"auto","created_at":"2025-12-04 09:57:30","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":38344,"visible":true,"origin":"","legend":"","description":"","filename":"renamed57d16.docx","url":"https://assets-eu.researchsquare.com/files/rs-8217702/v1/a50ecec7e9782cf1b376444f.docx"}],"financialInterests":"","formattedTitle":"Feline leukemia virus subgroup B uses phosphate transporters and shows promise as an envelope-based gammaretroviral vector for gene therapy in dogs","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGene therapy modifies genes by adding new genes, editing existing genes, or silencing problematic genes. There are two types of gene therapy: \u003cem\u003ein vivo\u003c/em\u003e gene therapy, in which a vector containing a gene is directly administered to the patient, and ex vivo gene therapy, in which a gene is introduced into cells taken from the patient and subsequently administered [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Research efforts are underway on various diseases, including cancer, viral infections, and genetic diseases [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Immunotherapy options used in humans have not yet been translated into veterinary medicine. A representative example is chimeric antigen receptor (CAR)-T cell therapy, which has recently shown good results in humans against hematopoietic malignant tumors and has been approved as a treatment for various B-cell malignant tumors and multiple myeloma [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, these advancements have limited benefits for canines [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].A preliminary study investigated the use of amphotropic murine leukemia virus (4070A MuLV) and gibbon ape leukemia virus (GALV) in canine tumor models [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Retroviral and lentiviral vectors are the primary gene transfer technologies used in CAR-T cell therapy. The introduced gene is maintained on the chromosome, even after cell division, via insertion into the chromosome. Replication-incompetent viruses are used for gene transfer and genome integration [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In this study, we focused on developing viral vectors using Feline Leukemia Virus subgroup B (FeLV-B). Considerable basic research has been conducted on FeLV-B infection. Viral antigens, antibodies against the virus, and viral genes can be detected using enzyme-linked immunosorbent assay and polymerase chain reaction (PCR), which have high sensitivity and specificity. A vaccine has also been developed against FeLV-B. FeLV-B is a promising and safe viral vector candidate for clinical studies [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFeLV is a gammaretrovirus commonly found in domestic cats worldwide [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. FeLV is associated with a high mortality rate in domestic cats and has been reported to induce malignant hematopoietic disorders, including lymphoma, myelodysplastic syndrome, acute myeloid leukemia, aplastic anemia, and immunodeficiency [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. FeLV subgroup A (FeLV-A) is the primary horizontally transmitted virus in domestic cats [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. FeLV-B results from recombination in the \u003cem\u003eenv\u003c/em\u003e region between FeLV-A and endogenous FeLV present in the feline genome [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. FeLV-B uses phosphate transporters 1 (Pit1) and 2 (Pit2) as entry receptors [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn addition to FeLV-B, various other viruses, such as koala retrovirus (KoRV; koalas), Gibbon ape leukemia virus (GaLV; Gibbons), Hervey pteropid gammaretrovirus (HPG; bats), 4070A amphotropic murine leukemia virus (4070A MuLV; murine), 10A1 MuLV (murine), and woolly monkey virus (WMV; monkeys), use Pits as viral receptors and can spread to hosts. Interspecies transmission may also occur [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Only a few cases of retroviral infections have been reported in dogs. \u003cem\u003eIn vivo\u003c/em\u003e infection in dogs showed the same result: no infection by the feline endogenous retrovirus RD-114 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Notably, cultured dog cells are susceptible to retroviral infections, such as RD-114 and 4070A MuLV [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Although FeLV-B infects canine cell lines [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], there have been no reports of FeLV-B receptors in dogs. In this study, we investigated the efficiency of FeLV-B Env-pseudotyped virus vectors for gene transfer in canine cells compared with GaLV, KoRV, HPG, and WMV Env-pseudotyped viruses. To ensure the safety of gene transfer methods using retroviral vector systems, it is necessary to identify retroviral receptors.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eIsolation of canine Pit (canPit)1 and 2 and construction of the expression vector\u003c/h2\u003e\u003cp\u003eRNA was isolated from canine peripheral blood mononuclear cells (PBMCs) and canine mammary tumors (KwDM) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] using the RNeasy kit (Qiagen, Hilden, Germany), and the extracted RNA was treated with recombinant DNase I (RNase-free; Takara, Shiga, Japan). Complementary DNA (cDNA) was synthesized using the PrimeScript II First-Strand cDNA Synthesis Kit (TaKaRa, Shiga, Japan) with oligo(dT) primers. canPit1 cDNA was amplified (based on the predicted sequence; XM_038691442.1) by PCR using the first primer pair Fe-923S and Fe-943R. canPit2 cDNA was amplified (based on the predicted sequence; XM_038689720.1) by PCR using the first primer pair, Fe-924S and Fe-944R (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). PCR products were digested with BamH1 and Xho1 for canPit1 and EcoR1 and BglII for canPit2, and each fragment was inserted into the pMSCVneo retroviral vector (TaKaRa). KOD-ONE Blue polymerase (Toyobo, Osaka, Japan) was used for PCRs according to the manufacturer\u0026rsquo;s instructions. The resulting plasmids were confirmed by sequencing (Fasmac Corporation, Atsugi, Japan).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell lines\u003c/h3\u003e\n\u003cp\u003eIn this study, we used the following cell lines: canine hepatocellular carcinoma (HCC) cells, canine mammary (KwDM) cells [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], \u003cem\u003eMus dunni\u003c/em\u003e tail fibroblast (MDTF) cells [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], GPLac (an env-negative packaging cell line containing the MuLV gag-pol gene and beta-galactosidase [LacZ]-coding pMXs retroviral vector) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], MDTF cells expressing canPit1 (MDTF-canPit1), and MDTF cells expressing canPit2 (MDTF-canPit2). Cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal calf serum and 1X penicillin\u0026ndash;streptomycin and subsequently incubated in a CO\u003csub\u003e2\u003c/sub\u003e incubator at 37\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eDetection of canPit1 and canPit2 expression levels via RT-qPCR\u003c/h3\u003e\n\u003cp\u003eRNAs from canine PBMC, KwDM, and HCC cell lines were extracted as described in the section \u0026ldquo;Isolation of canPit1 and canPit2\u0026rdquo; above. Briefly, cDNA was amplified using SYBR Premix Ex Taq II (Tli RNase H Plus; Takara) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). canPit1 was amplified using primers DA333046-F and DA333046-R. canPit2 was amplified using primers DA147288-F and DA147288-R and the internal control canine glyceraldehyde-3-phosphate dehydrogenase was amplified using the primers Fe-1011S and Fe-1025R as previously described [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Thermal cycling was performed according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003ch3\u003eEstablishment of cell line expressing canPit1 and canPit2\u003c/h3\u003e\n\u003cp\u003ePLAT-A (amphotropic MuLV) packaging cells were transfected with expression vectors (pMSCVneo-canPit1, pMSCVneo-canPit2, pMSCVneo-huPit1, or pMSCVneo empty vector) using TransIT\u0026reg;-293 reagent (Takara). PLAT-GP packaging cells were co-transfected with MuLV 10A1 and pMSCVneo-huPit2 to establish the huPit2 cell line. After 2 days, the supernatants were collected, filtered through a 0.22 \u0026micro;m filter, and used to inoculate MDTF in the presence of polybrene (10 \u0026micro;g/mL; Nakarai). The cells were cultured in a medium containing 600 \u0026micro;g/mL neomycin (G418) for 2 weeks. These cells were termed MDTF-canPit1, MDTF-canPit2, and MDTF-empty.\u003c/p\u003e\n\u003ch3\u003eIsolation of Env FeLV-B16\u003c/h3\u003e\n\u003cp\u003eBriefly, FeLV-B16 was isolated from 293T/ON-T #3 cells [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] using specific primers Fe-227S and Fe-919R. Thereafter, the amplicon was cloned into the TOPO vector. The plasmid was sequenced by Fasmac Corporation (Atsugi, Japan). Env was amplified using Fe-944S and Fe-958R based on the FeLV-B/B16 proviruses in the TOPO vector and cloned into pFUΔss between the \u003cem\u003eBam\u003c/em\u003eHI and \u003cem\u003eEco\u003c/em\u003eRI restriction sites. Env expression vectors were constructed using KOD-ONE Blue (Toyobo, Osaka, Japan) according to the manufacturer\u0026rsquo;s protocol. The resulting Env expression plasmids were confirmed by sequencing (Fasmac Corporation).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eEnv-pseudotyped virus preparation\u003c/h2\u003e\u003cp\u003eEnv-pseudotyped viruses carrying LacZ as a marker were prepared as previously described [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Briefly, GPLac cells were seeded in a six-well plate at a concentration of approximately 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/well 1 day prior to transfection. Notably, the Env expression plasmids used for viral preparation were pFUΔss GaLV Env, pFUΔss KoRV Env (opt-KoRV-A2), pFUΔss HPG Env (opt-HPG), pFUΔss WMV V655 Env [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], pFUΔss FeLV-B/GA, pFUΔss FeLV-B/ON-T, pFUΔss FeLV-B/MZ40-5B, pFUΔss FeLV-B/KG20-5B, pFUΔss FeLV-B/FO36-5B [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], pFUΔss FeLV-A/clone33 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], FeLV-B/B16, opt-KoRV-A2 Env, opt-HPG, opt-WMV V655 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], MuLV 10A1, and 4070A Env (Novus Biologicals). At 48 h after transfection using TransIT-293 transfection reagent, the culture supernatants were collected, filtered through a 0.22-\u0026micro;m filter or centrifuged at 15,400 \u0026times;g for 2 min at 4\u0026deg;C, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C as virus stock for further experiments.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eViral titer of Env-pseudotyped virus\u003c/h3\u003e\n\u003cp\u003eViral supernatants were collected from virus-producing cell lines (see the Env-pseudotyped virus preparation section) and centrifuged to remove cells and debris. Genomic viral RNA was purified from a small aliquot of the supernatant using the NucleoSpin RNA Virus Kit (Takara). RNA was then treated with DNase I to remove any residual plasmid DNA that may have been carried over from the transient transfection of packaging cells. Viral RNA samples were subjected to quantitative reverse transcription PCR (qRT-PCR) to determine the viral titer using Retro-XTM qRT-PCR (Takara) following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003ch3\u003eViral infection assay\u003c/h3\u003e\n\u003cp\u003eTarget cells were seeded in 24-well plates at a concentration of 1\u0026ndash;3 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well 1 day prior to infection. Thereafter, the cells were infected with 250 \u0026micro;L of Env-pseudotyped virus in the presence of 10 \u0026micro;g/mL of polybrene (Nacalai Tesque, Kyoto, Japan) for 2 h. After adding fresh medium, the cells were cultured for 2 days post-infection. After incubation for 48 h, the supernatants were removed, and the cells were fixed with 250 \u0026micro;L of 2% glutaraldehyde for 15 min at room temperature (20\u0026ndash;25\u0026deg;C). Thereafter, the cells were stained with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, and blue-stained nuclei were counted under a microscope. Viral titers are presented as infectious units (IU) per mL, with standard deviations. MDTF cells expressing only the vector were used as negative controls.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eVirus recombination analysis\u003c/h2\u003e\u003cp\u003eBriefly, recombination events were assessed using RNA-seq data obtained from HCC and KwDM cell lines infected with FeLV-B. Reads were trimmed using Trimmomatic (v0.39) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] to remove adaptors and low-quality sequences. Thereafter, the trimmed reads were aligned to a combined genome of \u003cem\u003eCanis lupus\u003c/em\u003e (GCA_011100685.1) and FeLV-B (LC861716) using HISAT2 (v2.2.1) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Alignments were coordinate-sorted and indexed using SAMtools (v1.22.1) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePhylogenetic and sequencing analysis\u003c/h2\u003e\u003cp\u003eA phylogenetic tree was constructed using the sequences listed in the Accession Numbers section. MUSCLE was used to align amino acid sequences for phylogenetic analysis [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and all other procedures were performed using the MEGA11 software package [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. For the phylogenetic analysis of Pit1, Pit2, and related proteins of gammaretrovirus receptors, the neighbor-joining method [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] was employed to construct a phylogenetic tree with amino acid substitutions in the JTT model [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The robustness of the trees was evaluated by bootstrapping 1,000 times [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eIsolation and characterization of canPit1 and canPit2\u003c/h2\u003e\u003cp\u003eIn this study, we investigated canPit1 and canPit2 as mammalian retroviral receptors. Pit1 and Pit2 are members of the solute carrier protein families SLC20A1 and SLC20A2, respectively. Initially, we isolated canine cDNA from canine KwDM cells and PBMCs using qRT-PCR, which were referred to as the predicted canPit1 (XM_038691442.1) and canPit2 (XM_038689720.1), respectively. Molecular cloning of PCR-positive samples was performed to obtain the canPit1 and canPit2 open reading frames. Our isolates and predictions for canPit1 and canPit2 in the database were 100% identical. Alignments of the amino acid sequences of the proteins encoded by the isolated canPit1 and canPit2 sequences are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. canPit1 and canPit2 were predicted to encode proteins with 687 and 653 amino acids, respectively, with approximately 89.2% similarity. Additionally, we conducted a phylogenetic analysis comparing our canPit1 and canPit2 clones to related sequences of Pit1 and Pit2 in other species and found that canPit1 and canPit2 receptors were classified in the same group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eExpression of canPit1 and canPit2 in canine tissues and cell lines\u003c/h2\u003e\u003cp\u003eIn the present study, we evaluated the expression of canPit1 and canPit2 in KwDM cells, HCC cells, and PBMCs. Notably, all samples exhibited canPit1 and canPit2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Although canPit2 was barely expressed in KwDM cells, CanPit1 and canPit2 were highly expressed in HCC cells. Additionally, canPit1 and canPit2 showed similar expression levels in PBMCs. Overall, these findings indicate that canPit1 and canPit2 are widely expressed in canine tissues and cell lines.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eEvaluation of canPit1 and canPit2 in retroviral infection\u003c/h2\u003e\u003cp\u003eIn this study, we conducted infection assays by infecting canine cell lines (KwDM and HCC) with FeLV-B Env-pseudotyped viruses and non-feline mammalian retroviruses (GaLV, KoRV-A, HPG, and WMV). To ensure that we used the same viral titer, we assessed the titer of the Env-pseudotyped virus (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). As expected, all Env-pseudotyped viruses could infect KwDM and HCC cell lines with FeLV-B/B16 and exhibited high viral titers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAdditionally, we investigated whether the infection required canPit1 or canPit2. MDTF cells were used because they are resistant to viral infections. MDTF-canPit1 and MDTF-canPit2 cells were constructed and used as target cells. Importantly, MDTF-canPit1 cells were permissive to all Env-pseudotyped viruses tested. The viral titers for Env-pseudotyped viruses varied (1,000\u0026ndash;100,000 IU/mL), with FeLV-B Env-pseudotyped viruses exhibiting high viral titers and WMV V66 showing the lowest viral titers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In contrast, MDTF-canPit2 was only permissive to FeLV-B/(ON-T and B16), GaLV, KoRV-A, and HPG, but not to FeLV-B (MZ40-5B, KG20-5B, and FO36-5B) and WMV V655. Notably, GaLV showed highly permissive infectivity, with viral titers reaching 10,000 IU/mL. MDTF cells expressing only the vector (negative control) were not infected with Env-pseudotyped viruses. FeLV-B/ (GA, MZ40-5B, and KG20-5B) and WMV V655 could only use canPit1, whereas FeLV-B (ON-T and B16), GaLV, KoRV-A, and HPG could use both canPit1 and canPit2 for infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Overall, our results suggest that FeLV-B (ON-T and B16) has the highest infectivity for canPit1 and canPit2.\u003c/p\u003e\u003cp\u003eIn addition, the pseudotyped FeLV-B/B16 did not infect KwDM or HCC cells infected with FeLV-B/GA. Conversely, FeLV-B/GA did not infect KwDM or HCC cells infected with FeLV-B/B16 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and D). Collectively, these results suggest that blocking the canPit1-mediated infection pathway prevents FeLV-B/B16 infections, highlighting the role of the pathway in FeLV-B/B16 infection.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eFeLV-B/B16 recombination analysis\u003c/h2\u003e\u003cp\u003eTo investigate whether FeLV-B/B16 can recombine with the canine genome, RNA-seq reads from HCC and KwDM cell lines infected with FeLV-B/B16 provirus were aligned to a combined \u003cem\u003eC\u003c/em\u003e. \u003cem\u003elupus\u003c/em\u003e and FeLV-B genome. Notably, the overall alignment rates were 95.86% (HCC) and 94.91% (KwDM). Additionally, the FeLV-B genome showed strong transcription, with 627,648 and 918,306 mapped reads in HCC and KwDM, respectively. However, split-read analysis from SAM supplementary alignments revealed no virus\u0026ndash;host (FeLV\u0026harr;dog) junctions and no intra-viral (FeLV\u0026harr;FeLV) rearrangements within the detection thresholds (\u0026ge;\u0026thinsp;3 unique split reads, mapping quality scores (MAPQ)\u0026thinsp;\u0026ge;\u0026thinsp;20, breakpoint concordance within \u0026plusmn;\u0026thinsp;5 bp) (32\u0026ndash;34), indicating that recombination between FeLV-B and the canine genome did not occur.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn humans, immunotherapy is widely used to treat various diseases, most notably cancer and autoimmune diseases. Developments such as tumor-specific CAR-T cells have broadened treatment options for human diseases, including blood cancers, specifically certain lymphomas, leukemias, and multiple myeloma [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. However, animals, particularly canines, have not benefited from these advancements. The retroviral system is the main method used in gene transfer technologies for CAR-T cell therapy, similar to the lentiviral system. In this study, we propose an alternative retroviral system for delivering gene targets using feline retrovirus (FeLV-B). FeLV-B has been extensively characterized and investigated for its role in the pathogenesis of viral infections, implying that it is a promising and safe viral vector for use in therapy.\u003c/p\u003e\u003cp\u003eMany retroviral envelope proteins bind to membrane transporters to enter the host for infection, including THTR1, which is used by KoRV-B [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and FeLV-A [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Pit1 has been described as an entry receptor for both GaLV and KoRVA [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. This has sparked research interest in their potential to enable viral entry into immune cells. The expression patterns of various receptors on host cells enable viruses to infect cells at specific stages of activation and proliferation [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Additionally, we investigated FeLV-B infection and found a high titer in canine cells, with canPit1 and canPit2 as entry receptors for infection. FeLV-B/ON-T and FeLV-B/B16 have higher titers of GaLV infection, which is commonly used for gene therapy, than other gammaretroviruses [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. CAR-T therapy products, such as axicabtagene ciloleucel (Yescarta, Kite Pharma Inc.; FOSUN Kite) and brexucabtagene autoleucel (Tecartus, Kite Pharma, Inc.), use a gamma retroviral vector (a murine stem cell virus-based vector pseudotyped with a GaLV envelope) [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. KoRV-A pseudotyped lentiviral vectors have also been investigated for gene transfer into isolated immune cells [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Our findings revealed that FeLV-B/ON-T and FeLV-B/B16 had higher titers than KoRV-A and GaLV. To corroborate this finding, we evaluated the expression of canPit1 and canPit2 and found high expression levels in two cell lines (KwDM and HCC) and canine PBMCs. These results support the notion that FeLV-B uses canPit1 and canPit2 as entry receptors for infection. Moreover, no recombination occurred between FeLV-B/N16 and the canine genome, as demonstrated by the infected replication-competent virus in HCC cells. Collectively, our findings indicate that the gammaretroviral system is a promising alternative to gene or CAR-T cell therapy. FeLV-B, particularly FeLV-B/ON-T and FeLV-B/B16, are promising candidates.\u003c/p\u003e\u003cp\u003eIn conclusion, we propose the use of FeLV-B Env as a viral vector system for gene therapy and demonstrate that FeLV-B Env-pseudotyped viruses can efficiently infect cells via Pit1 and Pit2. However, to investigate the cell introduction efficiency in more detail, their infectivity in newly isolated tumor cells and lymphocytes must be examined. Additionally, gene introduction using retroviral vectors carries the risk of carcinogenesis due to gene insertion into chromosomes, introduction into germ cells, and acquisition of proliferation ability in viral vectors [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Moreover, \u003cem\u003ein vivo\u003c/em\u003e safety could not be established in the present study. However, extensive basic research has been conducted on FeLV-B. Given that viral antigens and antibodies can be detected and a vaccine for FeLV-B has been developed [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], FeLV-B is expected to be a safe viral vector for therapy. However, the efficiency and safety of introducing viral vectors during \u003cem\u003ein vivo\u003c/em\u003e gene therapy in dogs and other animals must be examined in the future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eEthical approval\u003c/h2\u003e\u003cp\u003eAnimal studies were conducted in accordance with the guidelines for the care and use of laboratory animals provided by the Ministry of Education, Culture, Sports, Science, and Technology, Japan. All the experiments were approved by the Genetic Modification Safety Committee of Yamaguchi University.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis study was funded by the Japan Society for the Promotion of Science KAKENHI (Grant numbers: 23H02393 and 23K27086). KN received the funding. The funders had no role in the study design, data collection and interpretation, or the decision to submit the manuscript for publication.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e\u003cp\u003eKazuo Nishigaki conceptualized the study. Data curation was performed by Didik Pramono, Karin Inoue, Miharu Nishimoto, and Kazuo Nishigaki. Formal analysis of collected data was performed by Didik Pramono, Karin Inoue, and Kazuo Nishigaki. The funding for the study was acquired by Kazuo Nishigaki. The research/investigation was performed by Didik Pramono, Karin Inoue, Miharu Nishimoto, Loai AbuEed, and Kazuo Nishigaki. Research methodology was designed and implemented by Didik Pramono, Karin Inoue, Miharu Nishimoto, Loai AbuEed, Takuya Mizuno, Ariko Miyake, and Kazuo Nishigaki. Project administration and supervision were performed by Kazuo Nishigaki. Research validation was performed by Didik Pramono, Karin Inoue, Miharu Nishimoto, Ariko Miyake, and Kazuo Nishigaki. The original draft of the manuscript was written by Didik Pramono and Kazuo Nishigaki.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eWe are grateful to Dr. Masaharu Hisasue for HCC cells, Dr. Yoshinao Kubo for MDTF cells, and Dr. Toshio Kitamura for GP cells and the pMxs retroviral vector. This study was funded by the Japan Society for the Promotion of Science KAKENHI (Grant numbers: 20H03152 and 23K27086). KN received the funding. The funders had no role in the study design, data collection and interpretation, or the decision to submit the manuscript for publication.\u003c/p\u003e\u003ch2\u003eData availability statement\u003c/h2\u003e\u003cp\u003eThe accession numbers for publicly available data used in this study are as follows: Felis catus ASCT1 (NM_001278844), Felis catus ASCT2 (XM_045045413), gibbon Pit1 (XM_032176718), rat Pit1 (NM_031148), mouse Pit1 (NM_015747), human Pit1 (NM_005415), gibbon Pit2 (XM_032143780), rat Pit2 (NM_017223), and human Pit2 (NM_001257180). The sequences from the isolated samples described in this paper have been deposited in DDBJ/EMBL/GenBank under the accession numbers LC861716, LC886508, and LC886509. 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Vaccine 25:5531\u0026ndash;5539. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.vaccine.2006.12.022\u003c/span\u003e\u003cspan address=\"10.1016/j.vaccine.2006.12.022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8217702/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8217702/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChimeric antigen receptor (CAR)-T cell therapy, a cellular immunotherapy, has attracted considerable attention as a cancer treatment modality. Several CAR-T cell therapies have been approved for human use; however, this technology has limited application in veterinary medicine. Cancer is a notable health concern in dogs, with over 50% of dogs aged\u0026thinsp;\u0026gt;\u0026thinsp;10 years developing the disease. Viral vectors are useful for gene transfer and cellular protein expression, have high transfer efficiency, and can be expressed sustainably in the host. Particularly, retroviral vectors have received much attention because their genome can be introduced into the host genome, making them useful for delivering therapeutic genes into the host body. In this study, we investigated the efficiency of feline leukemia virus subgroup B (FeLV-B) Env-pseudotyped virus vectors for gene transfer compared with GaLV, KoRV, HPG, and WMV Env-pseudotyped viruses. Canine cells and cells expressing canine phosphate transporters (canPit1 or canPit2, entry receptors of FeLV-B) were infected with Env-pseudotyped viruses, and the infectious titer was measured. Additionally, cells that were persistently infected with retroviruses were identified, and receptor interference experiments were performed to investigate FeLV-B receptor utilization. Our findings revealed that FeLV-B/ON-T and FeLV-B/B16 Env-pseudotyped viruses exhibited notably higher infectious titers than other viruses. However, this study was limited to \u003cem\u003ein vitro\u003c/em\u003e infections using cell-based assays. Further investigations are required to determine the efficiency and safety of \u003cem\u003ein vivo\u003c/em\u003e gene transfers. Overall, this study highlights retroviral tropism in dogs and provides information on gene therapy systems.\u003c/p\u003e","manuscriptTitle":"Feline leukemia virus subgroup B uses phosphate transporters and shows promise as an envelope-based gammaretroviral vector for gene therapy in dogs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-04 09:57:25","doi":"10.21203/rs.3.rs-8217702/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2026-02-01T18:23:13+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-12-06T00:51:10+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-02T13:30:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-29T10:11:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Virology","date":"2025-11-26T23:08:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b66ec0e6-43a1-40e8-ac4a-32eaeed452c2","owner":[],"postedDate":"December 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-12T04:46:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-04 09:57:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8217702","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8217702","identity":"rs-8217702","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}