Replication-competent Retrovirus Research Articles

Abstract 7250: Serial assessment of GEN2, a retroviral vector for gene transfer of an optimized thymidine kinase and GM-CSF, for genomic integration into peripheral blood mononuclear cells (PBMCs) in a Phase 1 clinical trial in adult patients with solid tumors

Abstract GEN2 is a non-replicating retroviral gene transfer vector encoding a sequence-optimized herpes simplex thymidine kinase (HSV-TK), which activates the antiherpetic pro-drug ganciclovir (GCV), resulting in chain termination during DNA replication, triggering cancer cell apoptosis and release of neoantigens (NA). GEN2 also encodes hGM-CSF, which may augment antitumor immunity by enhancing NA presentation to immune effector cells. GEN2 in combination with valganciclovir (an oral formulation of GCV) was evaluated in a dose-escalating Phase 1 clinical trial in 61 adult patients (pts) with an advanced solid tumor (NCT04313868), with serial assessment of PBMCs to detect any GEN2 integration and replication competent retrovirus (RCR). Methods: PBMCs were obtained in Cycle 1 (following 1st/3rd dose), prior to and 2 hours after Cycle 2, and thereafter at 6-month intervals for 15 years, or until death or withdrawal of consent. Digital droplet PCR (ddPCR) for psi and envelope gene sequences in extracted genomic DNA from PBMCs was used to detect vector genome integration and replication competent retrovirus (RCR), respectively. Results: All 82 samples obtained from the intravenous cohorts 1-12 were below LOD (Limit of Detection) for RCR. All results for integration were BLOQ (Below Limit of Quantitation), whereas 4 samples collected at two hours following the end of infusion were above LOD, likely resulting from non-integrated cDNA. GEN2 continues to be studied in clinical trials in adult patients with solid tumors. Per the 2020 FDA Guidance for Industry (Long Term Follow-Up After Administration of Human Gene Therapy Products), serial assessment of vector integration is mandated throughout a pt’s participation in a gene therapy clinical trial and for 15 years following the last dose. These guidance documents were originally conceived when gene therapy was developed for treatment of rare hereditary diseases in pediatric pts; however, adult pts enrolled in early-stage clinical trials for cancer typically do not have a prolonged life expectancy (BMC Cancer 2011 11:426). In the context of proper and warranted usage of pts’ blood for testing, attention should be drawn to the limited expectation for a 2nd malignancy within the lifespan of this pt population, and the absence of detectable RCR in cancer patients receiving retroviral vectors over decades. A recent review noted no evidence of RCR in 1,595 post-treatment PBMCs obtained from 60 clinical trials (Mol Ther 2023 31(3):801). Our results confirm that GEN2 also shows negligible genotoxic risk for gene therapy of cancer at doses tested to date. Citation Format: Alison L. Hannah, Noriyuki Kasahara, Li Liu, Allen Wang, Joe McNulty, Robert G. Johnson. Serial assessment of GEN2, a retroviral vector for gene transfer of an optimized thymidine kinase and GM-CSF, for genomic integration into peripheral blood mononuclear cells (PBMCs) in a Phase 1 clinical trial in adult patients with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7250.

A recent gibbon ape leukemia virus germline integration in a rodent from New Guinea

Germline colonization by retroviruses results in the formation of endogenous retroviruses (ERVs). Most colonization's occurred millions of years ago. However, in the Australo-Papuan region (Australia and New Guinea), several recent germline colonization events have been discovered. The Wallace Line separates much of Southeast Asia from the Australo-Papuan region restricting faunal and pathogen dispersion. West of the Wallace Line, gibbon ape leukemia viruses (GALVs) have been isolated from captive gibbons. Two microbat species from China appear to have been infected naturally. East of Wallace's Line, the woolly monkey virus (a GALV) and the closely related koala retrovirus (KoRV) have been detected in eutherians and marsupials in the Australo-Papuan region, often vertically transmitted. The detected vertically transmitted GALV-like viruses in Australo-Papuan fauna compared to sporadic horizontal transmission in Southeast Asia and China suggest the GALV-KoRV clade originates in the former region and further models of early-stage genome colonization may be found. We screened 278 samples, seven bat and one rodent family endemic to the Australo-Papuan region and bat and rodent species found on both sides of the Wallace Line. We identified two rodents (Melomys) from Australia and Papua New Guinea and no bat species harboring GALV-like retroviruses. Melomys leucogaster from New Guinea harbored a genomically complete replication-competent retrovirus with a shared integration site among individuals. The integration was only present in some individuals of the species indicating this retrovirus is at the earliest stages of germline colonization of the Melomys genome, providing a new small wild mammal model of early-stage genome colonization.

Results from phase I/II study of NY-ESO-1-specific TCR gene-transduced T cell therapy (TBI-1301, mipetresgene autoleucel) in patients with advanced synovial sarcoma.

11558 Background: Synovial sarcoma is one of malignant soft tissue tumors with a poor prognosis. A cancer-testis antigen NY-ESO-1 is expressed in the tumors of 50-80% synovial sarcoma patients. The purpose of this study was to evaluate the safety and the efficacy of gene engineered autologous T cell product with NY-ESO-1 siTCR retroviral vector which expressed affinity-enhanced NY-ESO-1-specific TCR and siRNA to silence endogenous TCR (TBI-1301). Methods: This was an open label phase I/II study to evaluate safety, appearance of replication competent retrovirus (RCR), appearance of clonality, in vivo cell kinetics and clinical responses. TBI-1301 were manufactured from each subject’s leucocytes stimulated with anti-CD3 antibody and Retronectin and transduced with NY-ESO-1 siTCR retroviral vector. TBI-1301 was infused at split dose of 5 x 109 cells following cyclophosphamide treatment at 750 mg/m2 for two days to HLA-A*02:01 or HLA-A*02:06 positive subjects with synovial sarcoma expressing NY-ESO-1, which were surgically unresectable and refractory to anthracycline therapy. Results: Eight subjects were enrolled and treated with TBI-1301. The objective response rate was 50.0% with best overall partial response in 4 of 8 subjects according to RECIST v1.1/irRECIST and the median overall survival was 650 days. Cytokine release syndrome (CRS) occurred in 50.0% (4/8) and consisted of 1 subject with grade 1 CRS and 3 subjects with grade 2 CRS. All subjects who developed CRS recovered with prespecified treatment, in which 2 subjects were treated with symptomatic therapy, 1 subject was treated with tocilizumab and 1 subject was treated with both tocilizumab and corticosteroid. No subjects had immune effector cell- associated neurotoxicity syndrome (ICANS). RCR and clonal dominance were not detected in any subjects throughout the study period. Conclusions: Adoptive immunotherapy with TBI-1301 to selectively target NY-ESO-1 positive tumors will become a promising treatment for advanced or recurrent synovial sarcoma with acceptable toxicity. Clinical trial information: NCT03250325 .

A comparative study of human endogenous retrovirus <i>HERV-E λ 4-1</i> activation in autoimmune pathology

Considering the presence of immunomodulatory properties of human endogenous retroviruses, namely (i) the ability to activate the innate immune response by HERVs nucleic acids; (ii) the antigenicity of transcriptionally competent endogenous retroviruses envelope protein molecule, which causes polyclonal activation of lymphocytes; (iii) the absence of HERVs expression and protein production in the thymus during the immune tolerance formation, which allows us to consider these proteins as autoantigens or neoantigens, it seemed relevant to investigate the association of replication-competent human endogenous retrovirus HERV-E λ 4-1 with course of some of autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus. The aim of this work was a comparative study of the human endogenous retrovirus HERV-E λ 4-1 activation frequency in blood mononuclear cells in multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, as well as in chronic nervous system non-progressive diseases and the degenerative-dystrophic disease of the musculoskeletal system. The peripheral blood mononuclear cells were isolated by the venous blood centrifugation on Ficoll density gradient of 1.078 g/cm3. Expression of the HERV-E λ 4-1 envelope gene was detected by reverse transcriptase polymerase chain reaction. It was found that the HERV-E λ 4-1 envelope gene expression frequency in the chronic non-progressive diseases of nervous system, as well as in degenerative-dystrophic joint disease, is comparable to the expression frequency in conditionally healthy individuals. However, the HERV-E λ 4-1 envelope gene expression frequency in autoimmune diseases significantly exceeded that in conditionally healthy individuals and in non-inflammatory diseases. The maximum values of expression frequency were observed in active multiple sclerosis, significantly higher than in systemic lupus erythematosus and rheumatoid arthritis in the acute stage. Moreover, the expression frequency in the remission stage of multiple sclerosis was significantly lower than in the acute stage of the relapsing-remitted course, as well as in the progredient course. Estimation of HERV-E λ 4-1 envelope gene expression frequency at different severity levels of multiple sclerosis revealed its maximum rates at III and IV-V severity levels, both in relapsing-remitting and progressive course of multiple sclerosis. Thus, activation of the human endogenous retrovirus HERV-E λ 4-1 is associated with the course of autoimmune diseases, namely multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus; it positively correlates with the activity and severity of multiple sclerosis.

Phase I-II study using DeltaRex-G, a tumor-targeted retrovector encoding a cyclin G1 inhibitor for metastatic carcinoma of breast

Background: Metastatic breast cancer is associated with a poor prognosis and therefore, innovative therapies are urgently needed. Here, we report on the results of a Phase I-II study using DeltaRex-G for chemotherapy resistant metastatic carcinoma of breast.Patients and Methods:Endpoints: Dose limiting toxicity; Antitumor activity. Eligibility: ≥18 years of age, pathologic diagnosis of breast carcinoma, adequate hematologic and organ function. Treatment: Dose escalation of DeltaRex-G 1-4 x 1011cfu intravenously thrice weekly x 4 weeks with 2-week rest period. Treatment cycles repeated if there is ≤ Grade 1 toxicity until disease progression or unacceptable toxicity. Safety: NCI CTCAE v3 for adverse events reporting, vector related testing. Efficacy: RECIST v1.0, International PET criteria and Choi criteria for response, progression free and overall survival.Results: Twenty patients received escalating doses of DeltaRex-G from 1 × 1011 cfu to 4 × 1011 cfu thrice weekly for 4 weeks with a 2-week rest period. Safety: ≥ Grade 3 treatment-related adverse event: pruritic rash (n = 1), no dose limiting toxicity, no replication-competent retrovirus, nor vector-neutralizing antibodies detected. No vector DNA integration was observed in peripheral blood lymphocytes evaluated. Efficacy: by RECIST v1.0: 13 stable disease, 4 progressive disease; tumor control rate 76%; by PET and Choi Criteria: 3 partial responses, 11 stable disease, 3 progressive disease; tumor control rate 82%. Combined median progression free survival by RECIST v1.0, 3.0 months; combined median overall survival, 20 months; 1-year overall survival rate 83% for Dose Level IV. Biopsy of residual tumor in a participant showed abundant CD8+ killer T-cells and CD45+ macrophages suggesting an innate immune response. Two patients with pure bone metastases had >12-month progression free survival and overall survival and are alive 12 years from the start of DeltaRex-G therapy. These patients further received DeltaRex-G + DeltaVax for 6 months.Conclusion: Taken together, these data indicate that 1) DeltaRex-G has a distinctively high level of safety and exhibits anti-cancer activity, 2) PET/Choi provide a higher level of sensitivity in detecting early signs of tumor response to DeltaRex-G, 3) DeltaRex-G induced 12- year survival in 2 patients with pure bone metastases who subsequently received DeltaVax immunotherapy, and 4) DeltaRex-G may prove to be a biochemical and/or immune modulator when combined with other cancer therapy/immunotherapy.

Exploring the Expression and Function of cTyro3, a Candidate Zika Virus Receptor, in the Embryonic Chicken Brain and Inner Ear.

The transmembrane protein Axl was proposed as an entry receptor for Zika virus (ZIKV) infection in vitro, but conflicting results from in vivo studies have made it difficult to establish Axl as a physiologically relevant ZIKV receptor. Both the functional redundancy of receptors and the experimental model used can lead to variable results. Therefore, it can be informative to explore alternative animal models to analyze ZIKV receptor candidates as an aid in discovering antivirals. This study used chicken embryos to examine the role of chicken Tyro3 (cTyro3), the equivalent of human Axl. Results show that endogenous cTyro3 mRNA expression overlaps with previously described hot spots of ZIKV infectivity in the brain and inner ear. We asked if ectopic expression or knockdown of cTyro3 influenced ZIKV infection in embryos. Tol2 vectors or replication-competent avian retroviruses were used in ovo to introduce full-length or truncated (presumed dominant-negative) cTyro3, respectively, into the neural tube on embryonic day two (E2). ZIKV was delivered to the brain 24 h later. cTyro3 manipulations did not alter ZIKV infection or cell death in the E5/E6 brain. Moreover, delivery of truncated cTyro3 variants to the E3 otocyst had no effect on inner ear formation on E6 or E10.

Replication competent retrovirus testing (RCR) in the National Gene Vector Biorepository: No evidence of RCR in 1,595 post-treatment peripheral blood samples obtained from 60 clinical trials

The clinical impact of any therapy requires the product be safe and effective. Gammaretroviral vectors pose several unique risks, including inadvertent exposure to replication competent retrovirus (RCR) that can arise during vector manufacture. The US FDA has required patient monitoring for RCR, and the National Gene Vector Biorepository is an NIH resource that has assisted eligible investigators in meeting this requirement. To date, we have found no evidence of RCR in 338 pre-treatment and 1,595 post-treatment blood samples from 737 patients associated with 60 clinical trials. Most samples (75%) were obtained within 1 year of treatment, and samples as far out as 9 years after treatment were analyzed. The majority of trials (93%) were cancer immunotherapy, and 90% of the trials used vector products produced with the PG13 packaging cell line. The data presented here provide further evidence that current manufacturing methods generate RCR-free products and support the overall safety profile of retroviral gene therapy.

Long-term safety and efficacy of gene-corrected autologous keratinocyte grafts for recessive dystrophic epidermolysis bullosa

BackgroundRecessive dystrophic epidermolysis bullosa (RDEB) is a rare, devastating blistering genodermatosis caused by mutations in the COL7A1 gene, which encodes for type VII collagen and is necessary for dermal-epidermal adhesion and integrity. Disease manifestations include severe and debilitating wounds, aggressive squamous cell carcinomas, and premature death; however, there are currently no approved therapies. This Phase 1/2a, open-label study evaluated the long-term efficacy and safety of gene-corrected autologous keratinocyte grafts (EB-101) for chronic RDEB wounds.MethodsAutologous keratinocytes were harvested from participants with severe RDEB, transduced with a retrovirus containing the full-length COL7A1 gene, and grown into 5 × 7 cm (35 cm2) sheets. Gene-corrected keratinocyte sheets were then transplanted onto chronic RDEB wounds present for ≥ 12 weeks.ResultsSeven adult participants with severe RDEB were grafted with six sheets each (42 total sheets) onto wounds and followed for a mean of 5.9 years (range 4–8 years). Long-term improvements in wound healing and symptoms were observed. At year five, 70% (21/30) of treated sites demonstrated ≥ 50% wound healing compared to baseline by investigator global assessment. No sites with ≥ 50% wound healing were painful or pruritic, compared to 67% (6/9) of sites with < 50% wound healing (p < 0.001) at year five. Grafts were well-tolerated throughout long-term follow-up. No serious adverse events related to treatment were reported over a mean of 5.9 years of follow-up. No persistent systemic autoimmunity against type VII collagen or replication-competent retrovirus infections were identified, and no participants developed squamous cell carcinomas related to treatment during long-term follow-up.ConclusionsTreatment with EB-101 appears safe and efficacious, and produces long-term improvements in wound healing, pain, and itch for RDEB patients. Results from the Phase 3 randomized controlled trial are forthcoming.Trial registrationClinicalTrials.gov, NCT01263379. Registered December 15, 2010. https://clinicaltrials.gov/ct2/show/NCT01263379

Food and Drug Administration Guidance on Design of Clinical Trials for Gene Therapy Products with Potential for Genome Integration or Genome Editing and Associated Long-Term Follow-Up of Research Subjects.

With the burgeoning growth of the gene therapy industry, the Food and Drug Administration (FDA) has produced various guidance documents intended to help gene therapy manufacturers design their preclinical testing and clinical trials to facilitate the process of obtaining marketing approval. Biosafety professionals and institutional biosafety committees (IBCs) with oversight of clinical trials or biopharmaceutical manufacturing stand to benefit from understanding how these guidance documents set the standard for writing the clinical research protocols that are reviewed by IBCs. Although the FDA guidance documents are typically meant for manufacturers (either pharmaceutical companies serving as research sponsors or investigators at academic institutions), much of the content is useful for biosafety professionals and IBCs during the IBC review process. This article specifically addresses guidance documents pertaining to gene therapy vectors capable of genomic integration, testing for replication competent retrovirus, genome editing, and long-term follow-up of research subjects.

Residues 140-142, 199-200, 222-223, and 262 in the Surface Glycoprotein of Subgroup A Avian Leukosis Virus Are the Key Sites Determining Tva Receptor Binding Affinity and Infectivity.

Subgroup A avian leukosis virus (ALV-A) invades cells through gp85-encoded surface glycoprotein (SU) via specifically recognizing the cellular receptor Tva. To identify the key residues of ALV-A SU that determine the Tva binding affinity and infectivity in DF-1 cells, a strategy of substituting corresponding residues of SU between ALV-A RSA and ALV-E ev-1 (using Tvb as the receptor) was adopted. A series of chimeric soluble gp85 proteins were expressed for co-immunoprecipitation (co-IP) analysis and blocking analysis of viral entry, and various recombinant viruses based on replication-competent avian retrovirus vectors containing Bryan polymerase (RCASBP) were constructed for transfection into DF-1 cells and measurement of the percentage of GFP-positive cells. The results revealed that the substitution of residues V138, W140, Y141, L142, S145, and L154 of host range region 1 (hr1), residues V199, G200, Q202, R222, and R223 of host range region 2 (hr2), and residue G262 of variable region 3 (vr3) reduced the viral infectivity and Tva binding affinity, which was similar to the effects of the −139S, −151N, −155PWVNPF, −201NFD, Δ214–215, and −266S mutations. Our study indicated that hr1 and hr2 contain the principal receptor interaction determinants, with new identified-vr3 also playing a key role in the receptor binding affinity of ALV-A.

Current Perspectives in Human T-Cell Leukemia Virus Type 1 Infection and Its Associated Diseases.

Human T-cell leukemia virus type 1 (HTLV-1) is a replication-competent human retrovirus associated with two distinct types of diseases: a malignancy of mature CD4+ T cells called adult T-cell leukemia-lymphoma (ATL) and a chronic inflammatory central nervous system disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). It was the first human retrovirus ever associated with a human cancer. Although most HTLV-1-infected individuals remain asymptomatic for life, a subpopulation develops ATL or HAM/TSP. Although the factors that cause these different manifestations of HTLV-1 infection are not fully understood, accumulating evidence suggests that the complex virus-host interactions, as well as the host immune response against HTLV-1 infection, appear to regulate the development of HTLV-1-associated diseases. This review outlines and discusses the current understanding, ongoing developments, and future perspectives of HTLV-1 research.

Long-term follow-up for the development of subsequent malignancies in patients treated with genetically modified IECs

AbstractSubsequent malignancies are well-documented complications in long-term follow-up of cancer patients. Recently, genetically modified immune effector (IE) cells have shown benefit in hematologic malignancies and are being evaluated in clinical trials for solid tumors. Although the short-term complications of IE cells are well described, there is limited literature summarizing long-term follow-up, including subsequent malignancies. We retrospectively reviewed data from 340 patients treated across 27 investigator-initiated pediatric and adult clinical trials at our center. All patients received IE cells genetically modified with γ-retroviral vectors to treat relapsed and/or refractory hematologic or solid malignancies. In a cumulative 1027 years of long-term follow-up, 13 patients (3.8%) developed another cancer with a total of 16 events (4 hematologic malignancies and 12 solid tumors). The 5-year cumulative incidence of a first subsequent malignancy in the recipients of genetically modified IE cells was 3.6% (95% confidence interval, 1.8% to 6.4%). For 11 of the 16 subsequent tumors, biopsies were available, and no sample was transgene positive by polymerase chain reaction. Replication-competent retrovirus testing of peripheral blood mononuclear cells was negative in the 13 patients with subsequent malignancies tested. Rates of subsequent malignancy were low and comparable to standard chemotherapy. These results suggest that the administration of IE cells genetically modified with γ retroviral vectors does not increase the risk for subsequent malignancy.

Long-Term (≥4 Year and ≥5 Year) Overall Survival (OS) By 12- and 24-Month Event-Free Survival (EFS): An Updated Analysis of ZUMA-1, the Pivotal Study of Axicabtagene Ciloleucel (Axi-Cel) in Patients (Pts) with Refractory Large B-Cell Lymphoma (LBCL)

Long-Term (≥4 Year and ≥5 Year) Overall Survival (OS) By 12- and 24-Month Event-Free Survival (EFS): An Updated Analysis of ZUMA-1, the Pivotal Study of Axicabtagene Ciloleucel (Axi-Cel) in Patients (Pts) with Refractory Large B-Cell Lymphoma (LBCL)

The Oldest Co-opted gag Gene of a Human Endogenous Retrovirus Shows Placenta-Specific Expression and Is Upregulated in Diffuse Large B-Cell Lymphomas.

Vertebrate genomes contain endogenous retroviruses (ERVs) that represent remnants of past germline infections by ancient retroviruses. Despite comprising 8% of the human genome, the human ERVs (HERVs) do not encode a replication competent retrovirus. However, some HERV genes have been co-opted to serve host functions, most notably the viral envelope-derived syncytins involved in placentation. Here, we identify the oldest HERV intact gag gene with an open reading frame, gagV1. Its provirus contains an intact env, envV1, and the first open reading frame found in an HERV gag leader, pre-gagV1, which encodes a novel protein. This HERV is linked to a related gag gene, gagV3, and these three genes all show patterns of evolutionary conservation in primates. gagV1 and pre-gagV1 orthologs are present in all simian primate lineages indicating that this HERV entered the germline of the common simian primate ancestor at least 43 Ma, whereas gagV3 is found in Old and New World monkeys. gagV1 and gagV3 have undergone recurrent gene conversion events and positive selection. Expression of gagV1, gagV3, and pre-gagV1 is restricted to the placenta in humans and macaques suggesting co-option for placenta-specific host functions. Transcriptomic analysis of human tumors also found upregulated levels of gagV1 transcripts in diffuse large B-cell lymphomas. These findings suggest that these HERV-V genes may be useful markers for the most common type of non-Hodgkin's lymphoma and that they may have contributed to the successive domestications of env and gag genes in eutherians involved in the ongoing ERV-driven evolution of the placenta.

Establishment of a novel stable human suspension packaging cell line producing ecotropic retroviral MLV(PVC-211) vectors efficiently transducing murine hematopoietic stem and progenitor cells

Retroviral vectors derived from murine leukemia virus (MLV) are amongst the most frequently utilized vectors in gene therapy approaches such as the genetic modification of hematopoietic cells. Currently, vector particles are mostly produced employing adherent viral packaging cell lines (VPCs) rendering the scale up of production laborious, and thus cost-intensive. Here, we describe the rapid establishment of a human suspension 293-F cell line derived ecotropic MLV VPC. Using transposon vector technology, a packaging and envelope expression cassette as well as a transfer vector facilitated the establishment of a stable VPC yielding high titers of up to 5.2 × 106 transducing units/mL (TU/mL). Vectors were concentrated using ultrafiltration devices and upon one freeze-thaw-cycle still routinely yielded titers of > 1 × 106 TU/mL. Formation of replication-competent retroviruses was not detected. However and as a first generation transfer vector was used in this proof-of-concept (POC) study, gag gene sequences were transduced into target cells within a range of 1–10 copies per 1000 genomes indicating the homologous recombination of packaging construct elements with the transfer vector. High yield VPC vector productivity was stable over a couple of months and unintended integration of the transposase gene was not observed. Ecotropic MLV vector particles were demonstrated to efficiently transduce primary murine hematopoietic stem and progenitor cells. This novel concept should foster the future establishment of suspension VPCs.

Heterologous avian system for quantitative analysis of Syncytin-1 interaction with ASCT2 receptor

BackgroundHuman Syncytin-1 is a placentally-expressed cell surface glycoprotein of retroviral origin. After interaction with ASCT2, its cellular receptor, Syncytin-1 triggers cell–cell fusion and formation of a multinuclear syncytiotrophoblast layer of the placenta. The ASCT2 receptor is a multi-spanning membrane protein containing a protruding extracellular part called region C, which has been suggested to be a retrovirus docking site. Precise identification of the interaction site between ASCT2 and Syncytin-1 is challenging due to the complex structure of ASCT2 protein and the background of endogenous ASCT2 gene in the mammalian genome. Chicken cells lack the endogenous background and, therefore, can be used to set up a system with surrogate expression of the ASCT2 receptor.ResultsWe have established a retroviral heterologous chicken system for rapid and reliable assessment of ectopic human ASCT2 protein expression. Our dual-fluorescence system proved successful for large-scale screening of mutant ASCT2 proteins. Using this system, we demonstrated that progressive deletion of region C substantially decreased the amount of ASCT2 protein. In addition, we implemented quantitative assays to determine the interaction of ASCT2 with Syncytin-1 at multiple levels, which included binding of the soluble form of Syncytin-1 to ASCT2 on the cell surface and a luciferase-based assay to evaluate cell–cell fusions that were triggered by Syncytin-1. Finally, we restored the envelope function of Syncytin-1 in a replication-competent retrovirus and assessed the infection of chicken cells expressing human ASCT2 by chimeric Syncytin-1-enveloped virus. The results of the quantitative assays showed that deletion of the protruding region C did not abolish the interaction of ASCT2 with Syncytin-1.ConclusionsWe present here a heterologous chicken system for effective assessment of the expression of transmembrane ASCT2 protein and its interaction with Syncytin-1. The system profits from the absence of endogenous ASCT2 background and implements the quantitative assays to determine the ASCT2-Syncytin-1 interaction at several levels. Using this system, we demonstrated that the protruding region C was essential for ASCT2 protein expression, but surprisingly, not for the interaction with Syncytin-1 glycoprotein.Graphical abstract

World practice of providing scientific advice on the development and authorisation of innovative medicines

Current challenges to healthcare, i.e. the emergence of new diseases, lack of therapies for known diseases and life-threatening conditions, identification of patients who do not respond to standard treatment, on the one hand, and the evolution of scientific understanding of disease processes, medicines, therapies, causes of treatment failures, and implementation in clinical practice of innovations related to molecular biology and genetic engineering, on the other hand, create conditions and opportunities for the development of innovative medicinal products. A relatively new class of medicines is based on human cells and tissues (the term used in Russian legislation is biomedical cell products, BCP). However, the inability to accurately predict the efficacy and financial rewards of such medicines for pharmaceutical companies, as well as significant labour and financial costs associated with their development and clinical use, hinder their entry into the market. The aim of the study was to analyse the foreign regulatory setting for the development and launch of human cell- and tissue-based products, as well as approaches of foreign regulatory authorities to scientific advice, which can be drawn upon by the Russian expert authority when providing advice to BCP developers. The paper summarises the results of analysis of regulations establishing the procedure for providing scientific advice by EU, USA, and Russian regulatory authorities, and analyses the advice provided for the human cell- and tissue-based products which are now authorised in the EU and USA. The analysis of advice provided by foreign regulatory authorities shows that the largest number of consultations were given for medicinal products based on genetically modified cells for the treatment of cancer and genetic diseases. The questions were mainly related to the contents of specifications for finished pharmaceutical products, safety evaluation, curtailing of preclinical studies due to the lack of relevant animal/disease models, the number of subjects and efficacy endpoints in clinical studies, assessment of the appearance of replication-competent retroviruses.

Outcomes with KTE-X19 in patients (pts) with relapsed/refractory (R/R) mantle cell lymphoma (MCL) in ZUMA-2 who had progression of disease within 24 months of diagnosis (POD24).

7547 Background: KTE-X19 is an autologous anti-CD19 CAR T-cell therapy approved in the US and EU for the treatment of R/R MCL. In the ZUMA-2 study of KTE-X19 in R/R MCL, the objective response rate (ORR) at a median 17.5-mo follow-up was 92% (67% complete responses [CR]; Wang et al. ASH 2020 #1120). Here, we report results in pts with or w/o POD24, an indicator of poor outcomes (Visco et al. Br J Haematol 2019). Methods: Eligible pts with R/R MCL underwent leukapheresis and conditioning chemotherapy followed by a single infusion of KTE-X19. Efficacy results are reported for the 60 treated pts with ≥1 y of follow-up (median 17.5 mo); safety results are presented for all 68 treated pts. Results: High-risk disease characteristics were common in pts with (n=33) and w/o POD24 (n=35), although pts with POD24 had higher tumor burden and lactate dehydrogenase (LDH) levels, and more had blastoid type MCL (Table). ORR in pts with (n=28) and w/o POD24 (n=32) was 93% and 91%, with CR rates of 61% and 72%. In pts with and w/o POD24, median progression-free survival (PFS) was 11.3 mo (range, 0.9–30.3) and 29.3 mo (range, 0–35.9). Medians for duration of response (DOR) and overall survival (OS) were not reached in either group. Most common Grade ≥3 adverse events (AEs) in pts with vs w/o POD24 were neutropenia (91% vs 80%), thrombocytopenia (61% vs 46%), and anemia (55% vs 51%); Grade ≥3 cytokine release syndrome (CRS) and neurologic events occurred in 9% vs 20% and 27% vs 34%, respectively. There were no cases of Grade 5 CRS, KTE-X19–related secondary cancers, or replication-competent retrovirus in either group. In pts with vs w/o POD24, median peak CAR T-cell levels and median area under the curve were 53.4 cells/µL (range, 0.2–2566) and 583.4 cells/µL (range, 1.8–27,743.6) vs 112.4 cells/µL (range, 0.2–2589) and 1588.3 cells/µL (range, 3.8–27,238.7); by 12 mo, B cells were detectable in 8/11 (73%) vs 7/15 pts (47%) in ongoing response. Conclusions: KTE-X19 provided a high CR rate across all pts, with median DOR and OS not reached. Pts with POD24 had more aggressive high-risk disease characteristics (tumor burden, LDH levels, and blastoid MCL) and generally lower CAR T-cell expansion and PFS vs pts w/o POD24. Earlier intervention with CD19-directed CAR T-cell therapy may benefit pts with MCL with known high-risk factors. Clinical trial information: NCT02601313. [Table: see text]

Investigating the Effects of Frizzed2 Mutations on Craniofacial Skeletal Development

During early embryonic development cells that form different organs such as the face, limbs, heart are unspecified. Cell-to-cell communication using various signalling mechanisms supports development of tissues and organs. My project concentrates on understanding cell signalling pathway involving Wingless-related proteins (WNTs) and their role in embryonic bone and cartilage development. There is a human genetic disease caused by mutations in eight WNT pathway genes called Robinow Syndrome (RS). The functional impacts of these gene mutations on skeletogenesis remain unexplored. RS patients present with facial abnormalities and short stature. The objective of my project is to investigate the functional impacts of Frizzled2 (FZD2), one class of receptor that binds to WNTs, on facial skeletal development using the chicken embryo model. There are two FZD2 variants and that cause RS, one in the extracellular ligand binding domain (FZD2P142L) and the other in the intracellular domain(FZD2G434V). We are using the chicken since early facial and limb morphogenesis is conserved with humans. Additionally, local transgenesis is possible. We hypothesize that the FZD2P142L variant will cause abnormal ligand-receptor binding or receptor dimerization and the FZD2G434V variant will interfere with Dishevelled recruitment thus altering signal transduction. We used avian replication-competent retroviruses containing human FZD2 genes or the GFP control virus. Virus was added to high density micromass cultures composed of frontonasal mass mesenchymal cells and were assessed for differentiation after 4, 6 and 8 days of culture. All cultures formed cartilage and started to mineralize as shown by Alcian blue and alkaline phosphatase staining. However, the amount of cartilage significantly decreased by 8 days for all viruses (between 35-53%). AlkPO staining increased in the GFP cultures but was significantly decreased by all FZD2 viruses. At 4 days, the FZD2 viruses increased cartilage condensations compared to GFP. However, at 6 days all viruses were equivalent in terms of proportion of cartilage in the cultures. By 8 days the trend had reversed and the FZD2 viruses significantly decreased chondrogenesis. We conclude that the three viruses wtFZD2 and the two variants are equivalent in their effects, initially stimulating and then inhibiting chondrogenesis. This indicates that the variants are still active rather than causing a complete loss-of-function. It is also possible that the variants are causing a gain-of-function since they behave similarly to over-expression of wtFZD2. This project will help unravel the complex mechanisms underpinning skeletal dysplasias affecting the face and other organs such as the limbs due to the common molecular markers and transcription factors.

Wnt Signaling Drives Correlated Changes in Facial Morphology and Brain Shape.

Canonical Wnt signaling plays multiple roles critical to normal craniofacial development while its dysregulation is known to be involved in structural birth defects of the face. However, when and how Wnt signaling influences phenotypic variation, including those associated with disease, remains unclear. One potential mechanism is via Wnt signaling’s role in the patterning of an early facial signaling center, the frontonasal ectodermal zone (FEZ), and its subsequent regulation of early facial morphogenesis. For example, Wnt signaling may directly alter the shape and/or magnitude of expression of the sonic hedgehog (SHH) domain in the FEZ. To test this idea, we used a replication-competent avian sarcoma retrovirus (RCAS) encoding Wnt3a to modulate its expression in the facial mesenchyme. We then quantified and compared ontogenetic changes in treated to untreated embryos in the three-dimensional (3D) shape of both the SHH expression domain of the FEZ, and the morphology of the facial primordia and brain using iodine-contrast microcomputed tomography imaging and 3D geometric morphometrics (3DGM). We found that increased Wnt3a expression in early stages of head development produces correlated variation in shape between both structural and signaling levels of analysis. In addition, altered Wnt3a activation disrupted the integration between the forebrain and other neural tube derivatives. These results show that activation of Wnt signaling influences facial shape through its impact on the forebrain and SHH expression in the FEZ, and highlights the close relationship between morphogenesis of the forebrain and midface.