Biochemistry and Molecular Genetics Library Project Dissertation PDF

Comparing Various Oncolytic Viral Therapies with an Optimised HSV-Tk approach for Soft Tissue Sarcomas Victor Stefanescu November 2023 Contents 1 Introduction 1 1.1 Rhabdomyosa...

Comparing Various Oncolytic Viral Therapies with an Optimised HSV-Tk approach for Soft Tissue Sarcomas Victor Stefanescu November 2023 Contents 1 Introduction 1 1.1 Rhabdomyosarcoma: Epidemiology, Aetiology, Subtypes and Treatment 1 1.2 Considering Alternate Therapeutics................... 2 2 Therapeutic Genes 6 2.1 Current Knowledge on Detailed GCV-induced Mechanism of Apopto- sis in HSV-Tk+ Cells........................... 6 2.2 HSV1-Tk as the ideal suicide gene.................... 7 2.3 Going deeper, which strain of HSV produces Tk with highest catalytic activity of GCV.............................. 7 2.4 Two mutagenic sites offer optimisation potential in the Tk gene... 9 2.5 Enhancing the Bystander Effect through Connexin Transfection... 10 2.6 Tumor-Specific Promoters........................ 12 3 Delivery Vehicles 14 3.1 Viral Vectors............................... 14 3.1.1 Lentivirus, GHT viral vector and RNA-based (retro-) viruses as viral vectors.......................... 15 3.1.2 Adenovirus - Viability as a DNA-based viral vector...... 16 3.2 Adenoviral Vectors and RMS-specific Gene Transduction Efficiency. 17 4 Pro-drug Choice 22 4.1 Ganciclovir................................ 22 4.2 Altering Pharmacokinetics of GCV................... 22 5 Summary 23 1  Abstract Building upon a discovery last century that viral infection can cause remission of can- cer, oncolytic virus therapeutics have been developed to target a range of tumors. The components within each therapeutic such as delivery vector, gene transfected, and prodrug analogue can be interchangeable. In this report we compare various OV approaches for the treatment of soft tissue sarcomas, predominantly rhabdomyosar- coma, in order to pick an ideal configuration that yields the highest selectivity and tumor shrinking ability. We are comparing a modest array of FDA-approved OV therapeutics, with what is purported as one of the best therapeutics for RMS: the Herpes Simplex Virus – Thymidine kinase Ganciclovir (HSV-Tk/GCV) suicide gene therapy, which will help us to determine the best approach for effective eradication of the most common pediatric tumor. Chapter 1 Introduction 1.1 Rhabdomyosarcoma: Epidemiology, Aetiology, Subtypes and Treatment Sarcomas are a rare group of tumours of a mesenchymal origin affecting the musculoskeletal system. Relatively low prevalence has allowed this cancer subtype to fly under the therapeutic radar, less than 1% of cancers are sarcomas, and they have an occurrence of 2-4 affected persons per 100,000. Sarcomas are more prevalent in children (7% of cancers in children) than in adults (1% of cancers), and divided into two categories: Soft Tissue Sarcomas (STS) and bone sarcomas , with STS occurring at a higher rate (incidence ratio of bone sarcoma : STS is 1:4) prompting novel limb-sparing treatments for STS. The most aggressive and common soft tissue sarcoma in children is Rhabdomyosar- coma (RMS), representing 50% of all STS tumours in the age range of 0-14yrs , in adults, 1% of all cancers are STS of which 50% are RMS. Likewise, RMS has an unsatisfactory response to chemo- and radiotherapy; Adult patients receiving both have a 31% median 5-year survival rate, and a 27% median- survival rate at 10 years. Adult response to treatment is also significantly lower relative to children (5-year median survival rate of 27% and 61% respectively), which we can infer to be due to late diagnosis past the age of 19. The inefficiency of treatment is mostly attributed to the highly metastatic nature of the disease, alongside tumour chemoresistance. The rarity of the tumour poses problems in evaluating its determinants, it is not thoroughly studied granted it’s low prevalence in oncology. When considering response to all three common clinical treatments of: surgical 1 resection, chemotherapy, and radiotherapy, the 10 year disease-free survival rate still does not extend past 40-41%. Furthermore we argue against the dramatic lifestyle effects such as the acute toxicities of chemo- and radiotherapy as modalities to eradicate the tumour, also in spite of the novel advancements for surgical limb- and tissue-sparing techniques in clinic. Another key report on children with the metastasized disease noted a 5-year survival range of between 15% - 90% increasing with favourable features (tumour size and location, histology etc... ), this report also orders severity of the disease by 5-year survival rates into: low (95%), intermediate (65%), and high (15%) , the low survival rates associated with intermediate-high severity, are also demanding of improved therapeutics. The two common subtypes of RMS, alveolar and embryonic, are separated by occurrence at different bodily sites, and at different ages. It’s worth noting that although embryonic RMS accounts for 75% of cases , there is an annual increase in incidence of 4.1% regarding its alveolar counter-type , which is an important consideration, alongside the lacking modalities mentioned, factoring into a need for safer, more selective ways of treating the tumour, such as gene engineering within cell therapeutics. 1.2 Considering Alternate Therapeutics Therapeutics can be derived that target two distinct genotypes of RMS: gene fusions between PAX3/7-FOXO1, and mutations of genes encoding components involved in key signalling pathways (Ras). Therapeutics targeting mutated pathway components require more genomic data, and thus far extensive genomic data is lim- ited to alveolar RMS (ARMS), as this subtype has the worst prognosis. Genome Wide Association Studies (GWAS) are sparse to non-existent which are required to build aetiologies, and overall genomic background for personalised therapeutics, as mutations in signalling pathways and their respective components vary, from person to person, and age to age. For example, the commonly described Ras signalling pathway which is targeted in cancer therapeutics, can be looked at in the context of RMS and how personalisation of therapeutics can be difficult; mutation of any Ras pathway member was found in 50% of FOXO-1 fusion negative cases from 641 patients , making a singular approach difficult to follow. A “one shoe fits all approach” such as suicide gene therapy, could be the solution 2 Figure 1.1: From the SEER Program Incidence of RMS by age and subtype (2000 - 2017), we see higher prevalence of embryonal at early ages (0-3) which decreases in prevalence. For alveolar prevalence is quite constant up to the age of 19. to broadly addressing several cancer types with minimal toxicity. There is progressive evidence for one particular suicide gene therapeutic - HSV1-Tk/GCV in treatment of RMS. The mechanism behind HSV1-Tk/GCV is relatively simple, and involves the conversion of the inactive GCV prodrug by the enzyme Thymidine kinase (Tk), into an active, potent drug that the cancer cell is chemosensitive to. The Tk gene is transduced into a cancerous cell, alongside systemic injection of the GCV prodrug. Tk gene-expressing cells produce a kinase (phosphorylase enzyme) which phosphorylates GCV to GCV monophosphate, then GCV diphosphate, and finally GCV triphosphate which competitively inhibits deoxyguanosine triphosphate (dGTP), that arrests and/or suppresses DNA replication within the cancer cell to end its life cycle. Despite their low incidence, sarcomas provide ideal candidacy for suicide gene therapies, such as the most described HSV-Tk/GCV therapeutic. We mentioned before the concern with acute toxicities when using chemother- apy, which GCV is labelled under, to treat paediatric tumours. However we later discuss current labours in genetic engineering which aim to lower the dosage of GCV 3 Figure 1.2: Simple diagram illustrating the bystander effect - transferrence of GCV-triphosphate to neighbour HSV-negative cells. by making its activator enzyme (Tk) more metabolically efficient. GCV is con- verted into GCV monophosphate (GCV-P) by Tk, GCV-P is then converted to GCV diphosphate (GCV-DP) by thymidylate kinase, and then finally GCV triphosphate (GCV-TP) by cellular kinase. One of the earliest tests of HSV-1 Tk/GCV was conducted in 1992: The gene was inserted into a retroviral vector and injected intratumorally for cerebral glioma in rats. Systemic GCV therapy ensued and the study showed tumour regression, alongside no systemic toxicity. We can recall the reason for no toxic effects, being that GCV is a poor substrate for host human Tk present in the cytosol, hence no GCV-mono, di-, or tri-phosphate can be yielded and there is no overall effect on DNA polymerisation. It’s important to note that apoptosis does not occur directly due to GCV’s activa- tion of an apoptotic system, rather due to a delay in the S and G2 phase, and then a 4 delay in the overall proliferation cycle. A more in-depth analysis highlights DNA breaks, Bcl-2 decline, and caspase-9 activation caused by GCV’s incorporation into the cell - this was discovered in a study transfecting the HSV-Tk gene into Chinese Hamster Ovary cells (CHO) which is discussed here. Sustained GCV exposure is needed for apoptosis , and GCV-induced apop- tosis in HSV-1 thymidine kinase expressing cells involves DNA breaks, Bcl2-decline, and caspase-9 activation. This process can occur independent of the p53/21 signalling pathway and therefore demonstrates versatility in treating tumours of a diverse genetic background. P53 however, is instrumental in CD95 receptor translo- cation to the cell membrane when the p53 pathway is normal and active in HSVTk+ cells, this relies on caspase 8 activation. Another surprising finding was that CD95 receptor translocation occurs without the CD95 ligand, this is the case for neuroblastoma cells but the same cannot be said across all cancer cell types. Caspase-9 forms a complex with Apaf-1 triggered by cytochrome c and dATP , which initiates a protease cascade, the cascade is interrupted when caspase 9 is depleted and caspase 3 is inactivated – from which we deduce that caspase 9 is an upstream initiator in the apoptotic pathway. We also consider that germline p53 mutations are frequent in early-onset RMS and are associated with cancer predisposition in some young individuals. Given its involvement, Caspase-9 offers a potential strategy to enhance treatment efficacy in p53 deficient cells. The components of the HSV-Tk/GCV therapeutic we aim to explain and improve from literature are as follows - Tk gene variants - how the enzyme can be perfected via genetic engineering, to yield highest prodrug affinity, and rate of pro-drug metabolism. Prodrug/analogue choice - Already determined to be Ganciclovir, although we deliberate on other structurally similar analogues (Acyclovir (ACV)) and state why they are not as viable, some discussion will also be made on how GCV can be tweaked for higher affinity towards Thymidine kinase, and reduced toxicity. Viral vector - Gene transfection requires high viral vector oncotropism (speci- ficity towards tumour cell-types), proliferation, monitoring and visualisation capability - so that we can track the gene within tissues to ensure it hones in on correct cancer cell-types. 5 Chapter 2 Therapeutic Genes 2.1 Current Knowledge on Detailed GCV-induced Mechanism of Apoptosis in HSV-Tk+ Cells A study modelling the apoptotic mechanism considers two limitations of the HSV- Tk/GCV therapy - low target specificity, low transfection, and transduction efficacy of target cells. GCV mode of action is also poorly misunderstood, and requires clarification as Tk’s counterpart, additional information on the best administration technique is also required which will be discuss in the ”Prodrug Choice” section. Sustained GCV exposure is needed for apoptosis , and GCV-induced apop- tosis in HSV-1 thymidine kinase expressing cells involves DNA breaks, Bcl2-decline, and caspase-9 activation. This process can occur independent of the p53/21 signalling pathway and therefore demonstrates versatility in treating tumours of a diverse genetic background. P53 however, is instrumental in CD95 receptor translo- cation to the cell membrane when the p53 pathway is normal and active in HSVTk+ cells, this relies on caspase 8 activation , and another surprising finding was that CD95 receptor translocation occurs without the CD95 ligand, this is the case for neu- roblastoma cells but the same cannot be said across all cancer cell types. Caspase-9 forms a complex with Apaf-1 triggered by cytochrome c and dATP, which initi- ates a protease cascade, the cascade is interrupted when caspase 9 is depleted and caspase 3 is inactivated – from which we deduce that caspase 9 is an upstream initiator in the apoptotic pathway. We also consider that germline p53 mutations are frequent in early-onset RMS and are associated with cancer predisposition in 6 young individuals. Given its involvement, Caspase-9 offers a potential strategy to enhance treatment efficacy in p53 deficient cells. 2.2 HSV1-Tk as the ideal suicide gene We inspect the gene (UL23) within HSV-1 that encodes thymidine kinase, and note that it has an open reading frame of 376 codons, little to no conservation between different herpes viruses, and is fully protected by S1 nuclease digestion following hybridisation to Tk mRNA. Despite the lack of overall conservation, there are approx 6 coding regions within the Tk gene which do show conservation, which we can target later in gene engineering approaches. The higher binding affinities and substrate specificities of HSV-1 Tk to nucleoside analogues, relative to other analogues, make it more suitable as a gene of purpose. Additionally, neither Human Tk or Human Herpes Virus Tk (HHV) are able to phosphorylate GCV, which is why other virus variants are used in study. Comparative studies between Acyclovir (ACV) and GCV have boasted the latter’s superiority in substrate affinity. When compared with ACV, GCV demonstrated a 30-fold decrease in Vmax/Km with HSV-1 Tk, and GCV monophosphate a 492-fold decrease in Vmax/Km with cellular enzmyes (GMP kinase) that convert it to GCV diphosphate. These relative increases in affinity and rate of substrate saturation meant that more inhibitory triphosphate could be yielded. 2.3 Going deeper, which strain of HSV produces Tk with highest catalytic activity of GCV Despite the HSV1-Tk gene being used in over 30 clinical gene therapy trials for various human cancers, engineering approaches are high in demand as the high dose of GCV required for complete tumour regression is immuno- and myelosuppressive. We consider two main limitations of using GCV as a prodrug - substantial toxicity, 7 and immunosuppressive action following high dosage , immunosuppressive action is also known to hamper the bystander effect. We are not looking to replace GCV, as an emulatory analogue is hard to come by, we instead look at mutant strains of HSV-1/2 that provide better catalytic activity, this allows us to reduce the dosage of GCV, hence lowering toxicity, sparing immune damage, and ensuring bystander effect happens at full potential. We already established that wild-type Tk shows a weak affinity for GCV (47um); a library of semi-random mutant strain enzymes was developed, and tested for im- provements in both GCV- and ACV-mediated tumor killing. The SR39 HSV-1 Tk mutant displayed the greatest kinetics with GCV relative to wild-type Tk (Km = 14- fold decrease). SR39 shows multiple other benefits, such as improved sensitivity when used as a PET reporter gene for visualisation and monitoring. The amino acid variation of SR39 is given below, and we will later disclose other mutations purported to optimise the Tk gene - The second mutant HSV-1 thymidine kinase to be considered is mutant 30 : examined in vitro for causality in cell sensitivity to GCV and in vivo by kinetic characterisation and molecular modelling, the results of which are shown below. With mutant 30 transduction we mostly see that rat C6 glioma cells exhibited a 2-3 log increased sensitivity to GCV compared to wild-type HSVTk and a 2.7-fold higher Tk expression. Additionally 3/5 mice injected with mutant 30 showed no visible tumour at the end of the experiment - ergo mutant 30 provided complete tumour ablation at much lower doses, and within a shorter time-frame of GCV administration. Finally we note that despite poor connexin-43 expression and limited gap junction communication in C6 cells, a significant reduction in tumor size occurred in mice with 20% mutant 30-expressing cells after GCV treatment. In contrast, no bystander effect was observed with wild-type TK. The noteworthy bystander effect with GCV at 20% mutant 30 expression underscores the potential impact of structural changes induced by mutant 30 in enhancing therapeutic outcomes. While mutant 30 shows much poorer Km values for thymidine and GCV, it demonstrates increased sensitivity to prodrugs in cell culture assays compared to cells expressing wild-type TK. The enhanced prodrug sensitivity in mutant 30 is attributed to its poor thymidine binding, resulting in a kinetic advantage despite higher Km values. The passage emphasizes that mutant 30’s distinct advantage lies in its increased specificity for prodrugs, suggesting its superior ability to catalyze phosphorylation in a complex nucleoside milieu. 8 2.4 Two mutagenic sites offer optimisation poten- tial in the Tk gene The sequences of sites 3 and 4 in Herpes Virus Thymidine kinase showed the highest participation in substrate binding , this was determined via comparing catalytic efficiencies of a library of mutagenic, functional clones with random sequences substi- tuted at said sites. For example a substitution of the random amino acids XYZ at site 1 which previously had ABC showing a fold-increase or decrease in substrate affinity (Kcat/Km) would mean sequence XYZ either hinders or facilitates function respectively. Site 3 is the most conserved and has a consensus sequence of DRH, site 4 follows merely 6 amino acids after and has the consensus sequence CYP - we are to estimate that the 6 amino acids in between also have some functionality in specificity towards substrate-binding. We use ED50 (median effective dose) to measure level of sensitivity of Tk to GCV, as this reading is defined as ”the dose of medication required to produce a response in 50% of the cell population administered” ), as we are looking for the the lowest possible non-toxic dose of GCV to administer, that will be enacted upon by the Tk enzyme for high yield of GCV-TP molecules. Mutants with poor phosphorylation ability towards thymidine kinase are also of interest, as loss of thymidine – thymidine kinase binding results in a decrease of com- petition between thymidine and nucleoside analogues (GCV). It is desired that the enzymes derived with the 3-6 amino acid alterations in the consensus sequences of site 3 and 4, enact a shift in specificity from thymidine to GCV, for direct therapeu- tic value (Fig) prompted by the competitive inhibition issue, that wild-type HSV1 preferentially phosphorylates thymidine instead of nucleoside analogues GCV and ACV which is mentioned prior, and the ratio of GCV/thymidine phospho- rylation by mutant 30 and 132 enzyme compared to wild type HSV1-Tk is 31- and 32-fold higher respectively. We can further decrease the inhibiting competition between thymidine and GCV by site-directed mutagenesis in the HSVTk consensus sequence between residues 54-65, as shown in a different study that a threonine-serine conversion at position 63 increased Km (decreased thymidine affinity) from 0.5um in wild-type Tk to 2.3 in the mutant enzyme Tk T62S. So far this gives us multiple avenues for site directed mutagenic optimisation of the HSV1-Tk gene for increasing affinity to the desired prodrug of choice. Analysis of altered residues preceding/N-terminal to the conserved tripeptide motifs of each site was aimed at determining contribution to binding pocket and hydrophobicity of the active site cavity. Out of 80 selected clones in this procedure, 10 showed significant enhancements in phosphorylation preference to- 9 Figure 2.1: Amino acid mutations in HSV-1 Tk residues 159-174 that yield different kinetics with GCV wards at lease one nucleoside analogue, subsequent assay analysis narrowed clones down to 3 for enhanced sensitivity to GCV and ACV, with one clone pCMV:75 demonstrating a GCV-sensitivity fold increase as large as 43. 2.5 Enhancing the Bystander Effect through Con- nexin Transfection Numerous suicide gene cancer therapy studies have established the importance of the bystander effect, in transmitting the metabolised prodrug (GCV-triphosphate) to nearby HSV-Tk negative cells. Here we give an overview of the role of connexin 26 (Cx26) in mediating the bystander effect , and determine whether skeletal muscle tumor cell lines, such as RMS, can exhibit substantial bystander effect mediated transference of GCV-triphosphate, for compensation of limited HSV-Tk gene transduction. This is important mostly because we can consider other thymidine kinase analogues (not just HSV1-derived), to increase bystander effect - for example human derived deoxycytidine kinase(A Neschadim), we also use evidence below to suggest Cx26 transduction as an adjunctive therapeutic with HSV-Tk/GCV. Transformation into a cancer cell type decreases expression of functional gap junctions, as seen in bladder cancer cell types. Focusing on two genes responsible for gap junction formation, and down-regulated during transformation - Connexin 43 and 26. 10  The bystander effect is a crucial component of HSVtk/GCV gene therapy, playing a pivotal role in enhancing therapeutic efficacy. This phenomenon is essential for overcoming gene transduction limitations and elicits an inflammatory immune response against tumors. Connexins (Cxs), specifically Cx26, Cx32, and Cx43, are integral in mediating the bystander effect. The downregulation of Cx ex- pression in transformed cells is associated with enhanced proliferation and reduced differentiation. In human bladder cancer cells, introducing Cx26 significantly extends the bystander effect in HSVtk/GCV gene therapy, indicating its potential as a diffuser or dose-reducing factor. Figure 2.2: Survival of Bladder Cancer Lines - mock infected (control), infected with virus alone, infected with virus + reporter gene, and infected with virus + Cx26 Furthermore, decreased Cx expression and intercellular communication are ob- served in human bladder cancer cells : Cx26, when introduced into bladder cancer cells, significantly enhances growth inhibition and decreases tumor volume, showcas- ing its tumor-suppressive effects. In HSVtk/GCV therapy for bladder cancer, the cytotoxicity produced by Cx26 complexes is statistically significant compared to con- trol complexes. In vivo studies with nude mice indicate a reduction in both tumor volume and incidence with Cx26 expression, highlighting an enhanced bystander effect. Research on myogenesis provides insights into connexin dynamics during muscle 11 development : Connexin43 (Cx43) is highly expressed in cycling myoblasts during early muscle development, forming functional gap junctions crucial for myoblast fusion. Understanding these dynamics is particularly relevant in the context of RMS, where disruptions in Cx43 dynamics during myogenesis may contribute to abnormal muscle cell development and disease progression. In the realm of RMS, it is crucial to consider the neoplasia mechanism caused by misregulated myoblast fusion. Additionally, the interaction between Pannexin 1 (PANX1) and Cx43 suggests a complex network of molecular events influencing RMS malignancy. These findings underscore the intricate nature of RMS de- velopment and its potential association with gap junction proteins. Furthermore, the expression of gap-junction proteins, particularly Connexin 43 (Cx43), varies between normal and malignant human tissues, providing insights into cancer cell behavior : Reduced expression of Cx43 in breast cancer implies a po- tential role in tumorigenicity. The heterogeneous distribution of connexins in human malignancy underscores the complexity of their functional regulation, offering valu- able information for improving therapies like HSVtk/GCV by manipulating connexin expression. These findings collectively emphasize the importance of gap-junction-mediated intercellular communication in maintaining tissue homeostasis, controlling growth and differentiation, and influencing therapeutic outcomes in diseases like bladder cancer and RMS. The detection of gap-junction proteins in various tissues highlights their diverse roles and potential implications for targeted therapies. The goal here is developing a recombinant viral vector carrying both the HSV-1 Tk gene alongside genes encoding for Connexin proteins specific to the RMS cell types, by which we would be able to not only phosphorylate GCV optimally, but also ensure maximum bystander cell transferrence of the metabolised prodrug, which we’ve explained relies heavily on intracellular functional gap junctions. 2.6 Tumor-Specific Promoters One approach in lowering chemotherapy dose of GCV is via introducing a myogenin promoter into HSV plasmids, with increased selectivity towards expressing HSV-Tk in ARMS cells, whilst sparing expression in unaffected cells : The myogenin pro- moter was modified by removing the NF1 or MEF3 motifs, resulting in enhanced specificity for ARMS cells (RH30) over healthy myogenic cells (C25). These modi- fied promoters exhibited reduced activity in differentiating C25 myoblasts and higher activity in proliferating C25 myoblasts, this also highlighted the regulatory role of NF1B in myogenin expression. The engineered LV-miniMg-MEF3/NF1-HSV pro- 12 moter effectively reduced ARMS cell viability in vitro and demonstrated significant efficacy in reducing ARMS tumor size in a mouse xenograft model, correlating with increased apoptosis. Combining LV-miniMg-MEF3/NF1-HSV suicide gene therapy with the HSV-TK/GCV system substantially reduced chemotherapy concentration (24-fold) while maintaining therapeutic efficacy. Overall we see how modifying differ- ent promoters within the HSV1 plasmid can lead to a higher quality HSV-Tk/GCV therapeutic. 13 Chapter 3 Delivery Vehicles 3.1 Viral Vectors We’ve already mentioned some viral vectors as a means of transfecting an optimal HSV1 Tk gene for the suicide gene therapeutic. Table 3.1: Tumor types currently under clinical trials for HSV-Tk/GCV therapeutic, with type of vector delivery Tumor Type/ Vector Clinical Stage Reference Cancer site Glioblastoma Liposoma I/II Prostate Adenoviral I Head/neck Adenoviral I Hepatocellular Adenoviral I Carcinoma Recurrent Adenoviral I Gynecologic Using the table created above, we can see the various delivery vectors used for different tumour types, and fixate on the trials that used adenoviral methods, as these are for tumours most similar RMS, we mentioned before that RMS can also arise in the prostate, head and neck areas. There are countless innovations to be attentive to, regarding suicide gene – pro- drug therapies, three of which are highlighted well are A) the improvement of viral 14 vectors for targeted gene delivery, using tissue specific promoters and receptors. B) Improvement of oncotropism, for selectivity and safety reasons. C) Combination with classical cancer treatments (radio-, chemo-, excision). We won’t delve into the latter, as complete replacement with standalone GDEPT forms the basis of our argument. 3.1.1 Lentivirus, GHT viral vector and RNA-based (retro-) viruses as viral vectors The foundation of our report is a trial for suicide gene therapy that used a lentiviral construct to deliver the HSV-1 Tk gene with relatively high success , retroviral vectors, especially lentiviruses, are commonly explored for delivering the HSV-Tk transgene in rhabdomyosarcoma. The GHT vector, genetically engineered in E. coli, demonstrates safety and transfection efficiency advantages over traditional agents like lipofectamine and PEI 25 kDa : It efficiently condenses pSR39 into nanoparti- cles (¡100nm), with in vitro studies confirming the GHT vector’s ability to deliver pSR39 to SKOV-3 cancer cells, and inducing significant cell death without observed toxicity in control groups. Long-term toxicity assessments via clonogenic assays support the vector’s potential by preventing cancer cell colony formation. Similar success is seen with lentiviral vectors, demonstrating stable transduction of HSV-Tk into rhabdomyosarcoma cell lines, emphasizing the efficiency of lentiviral plasmids for bicistronic expression. Lentiviral vectors derived from HIV-1 exhibit efficiency in transducing hepatocellular carcinoma (HCC) cells in vitro and in vivo, demonstrating strong antitumoral efficacy with minimal normal liver toxicity. The integration of woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sig- nificantly enhances retroviral vector gene transducing capability, underscoring the importance of post-transcriptional gene expression regulation in gene delivery vehi- cle construction. These collective findings highlight the promise and versatility of retroviral vectors, particularly lentiviruses, in advancing HSV-Tk-mediated gene therapy for various cancers. Recalling briefly what we can attribute to the success of lentiviral vectors in the HSV1-Tk/GCV therapeutic on Rh30 lines : we note that the lentiviral construct used is bicistronic, with each cistron separated by an internal ribosomal entry site (IRES), and the construct expresses GFP for monitoring as well as HSV-Tk. Ex- pression of transgenes is strongly regulated by the CMV promoter. Retrovirus vector delivery of HSV-Tk gene therapy has reported success in treat- ment of numerous cancer cell lines such as melanoma , lymphoma, and glioma cell lines. Generally speaking gene co-transfer efficiency of HSVTk + Lac (reporter) is 15 deemed satisfactory in retro-viral vectors (30% in a study for hepatocellular carci- noma HSVTk therapeutic): We show in hepatocellular carcinoma, that retrovirus delivered Tk in HSV-Tk/GCV therapy meets the same criteria as for all ubiquitous suicide gene therapies, such as significantly declining size of Tk+ BEL-7402 cells (hepatoma) upon GCV administration, and positive in vitro effects, it’s important here also to note a histological finding that also supports retroviral agent use in HSVTk delivery - such that we observe spread of retroviral particles, and infection of neighbouring BEL-7402, propagating efficient HSVTk transduction. However, In vivo application of retroviruses for suicide gene transfection are prob- lematic due to difficulty in high titre production, transfection of dividing cells only, and risk of insertional mutagenesis. Whereas the opposite can be said for ade- noviruses, which can produce high titre and offer little possibility of insertional mu- tagenesis. As well as the fact that, although retroviruses demonstrate power in infection of rapidly dividing cells, direct in situ inoculation of tumorous tissue commonly requires producer cells - such as stem cells , adenoviruses too require producer/packaging cells, for example mesenchymal stem cells , but this is less common. 3.1.2 Adenovirus - Viability as a DNA-based viral vector Studies validate Ad5 as a vector capable of infecting up to 90-100% in cancer cell types such as thoracic for mesothelioma: With transfection in vitro of 50-100 viral particles per tumour cell, this was shown using an Ad.RSVlacZ vector that carries a reporter gene (lacZ ) and HSVtk enzyme was observed in the nucleus and cytoplasm via immunohistochemical staining. Also in accordance was tumour cell sensitisation to much lower doses of GCV, in response to recombinant adenovirus vector carrying HSVtk (Ad.RSVtk), as IC50 was 3-4 logs lower in Ad.RSVtk compared to the control. A similar sensitisation was seen when this was performed on lung cancer cell lines, with the A549 cell line exhibiting a higher IC50. Occurrence of the bystander effect too was proven in this study when using Ad5 as a viral vector, the criteria for the bystander effect in this specific study being a 1:10 infected:uninfected ratio, or 10% of cells requiring infection for propagation of bystander effect. One of the main problems arising from the use of Ad5 as a vehicle is rapid clearance from circulation, meaning few of these oncolytic viruses reach the cancer site - this is attributed to Ad5’s tendency to bind to non-target cells and components following administration, such as erythrocytes, antibodies , and platelets. 16  One way in which we can optimise Ad5 gene transduction is detailed, from a study involving the engineering of a Ad5 plasmid with a Arginylglycylaspartic acid (RGD) motif on the prominent HI capsid surface loop , this followed identification of RGD being able to mediate CAR-independent docking of Ad5. The aim of this is to to hopefully transduct cells lacking CAR - an essential docking site, or receptor of Ad5 in cancer cells. This modification was performed in multiple studies to observe differences in gene transduction efficiency, and cancer cell regression of the pancreas , breast , ovary and, colon/rectum. Valuable data was gathered from testing this HSV-TK + Coli.NTR recombinant, fibre-modified Ad5 vector (Ad-RGD-hT-TK/NTR) on breast tumour models, and showed that transfer efficiency, cytotoxic effects, survival rate, inhibition of tumour growth and safety (measured by liver toxicity) was far improved when compared to controls. We’ve thus far scarcely discussed safety and risk management owing to each com- ponent of HSV-Tk/GCV. Data on the engineering of the HSV1-Tk therapeutic gene is lacking due to tunnel-vision on the immense regression of the tumour in each re- spective clinical case, and furthermore by the fact that the therapeutic already shows high oncotropism (selectivity towards cancer cells), and lack of destructive potential towards healthy neighbour tissue. In regards to delivery vehicle, it is important to consider whether there is a risk of Ad5 merging with DNA - this risk is measurable by detecting adenoviral DNA in blood or urine, using polymerase chain reaction (PCR) in intervals: This was done for Ad5 viral vector use for HSV-Tk/GCV treatment towards prostate cancer, alongside typical symptomatic overview of viral infection (fever, liver dysfunction) and reassured safety, mild clinical toxicity was observed after two cycles of the therapeutic. 3.2 Adenoviral Vectors and RMS-specific Gene Trans- duction Efficiency More than 500 clinical trials involving adenovirus as a vector (primarily Ad5) have been carried out, comprising 20% of all clinical trials within the domain of gene ther- apy, the prospect of which is to tailor high oncotropism towards lysis of tumour cells, which is usually followed by recruitment of immune cells to the tumour microenvi- ronment ; Vectors should replicate selectively and express their niche transgene over a sustained period of time, although a suicide gene in this case requires only transient expression owing to the relatively short clinical duration it takes to treat the cancer. 17  The linear, double stranded DNA molecules of adenoviruses are relatively medium- sized (26-45kb) compared to other DNA viruses. Stability of the Ad genome is dependent on its net size, as a genomes larger than the wildtype Ad5 undergo rearrangement at a faster pace, and are therefore more genetically unstable. Ad vectors with genome sizes of 105-106% relative to wildtype Ad can be easily gen- erated, though they lack stability , this has implications on the engineering of an optimal Ad plasmid for Tk gene transduction – as we rely on inclusion of a multitude of various different transgenes, promoters, enhancers and such for a HSV-Tk gene therapy that yields high selectivity, and potency for desired clinical outcomes. 57 subgroups of adenoviruses have been identified and categorized into 6 sub- groups (A-F) based on tropism and receptor expression , there is no indication of muscle-specific tropism in any of the subgroups. However, skeletal muscle suscep- tibility can be enhanced by transgenic over-expression of CAR. Muscle-specific over=expression of the adenovirus primary receptor CAR identifies Ad5 as the suggested serotype for therapeutic gene transduction, particularly aiming to address low transducibility in skeletal muscle types related to rhabdomyosarcoma. Notably, Ad37 exhibits superior transduction in rhabdomyocytes, showing promise along with Ad17. Genomic stability of Ad vectors is crucial, with larger genomes than wildtype Ad undergoing faster rearrangement, rendering them genetically unstable : While vectors with sizes of 105-106% relative to wildtype Ad can be generated easily, their lack of stability raises implications for constructing optimal Ad serotypes for Tk gene transduction, essential for achieving high selectivity and potency in HSV-Tk gene therapy. Efficient muscle-specific transgene expression reveals potential of replication- defective human adenovirus vectors for therapeutic gene delivery to skeletal muscle cells. Muscle-specific expression is achieved using a tissue-specific promoter/enhancer, exemplified by truncated (1.35 kb) muscle creatine kinase (MCK) or fast troponin I (TnI) promoter/enhancer. Furthermore, the Transient Chimeric Ad5/37 Fiber study introduces a novel miRNA-regulated method enhancing Ad vector transduction. The Ad5 vector, incor- porating a knob from Ad37, demonstrates improved transduction rates and efficient delivery to tumor cells via NK-92 carrier cells. This transiently capsid-modified chimeric vector holds promise for cancer and gene therapy, particularly in immune therapy involving natural killer cell-derived carriers. The optimal serotype rAAVrh.10 is what we focus on, rAAVrh.10 displays no- table muscle tropism following intraperitoneal delivery. It exhibits the highest trans- duction efficiency in skeletal muscle, accompanied by robust expression in the heart 18 and diaphragm. Minimal off-target transduction in other tissues and a favorable safety profile further endorse rAAVrh.10 as the best viral vector for HSV1-Tk/GCV therapeutic, and these collective insights provide information on tropism, stability and transduction efficiency when it comes to viral vectors as a delivery method. Efficient distribution of the toxic metabolised prodrug GCV to RMS cells has proven successful, owing to bystander effect mediation through gap junction inter- cellular communication, and kinetics between the HSV1-Tk enzyme and GCV are likewise not showing any hindrances, although we’ve managed to study these more in-depth to outline how affinity and reaction rate (phosphorylation velocity) can be enhanced. This leads us now to discuss the core issue surrounding clinical translation of viral vector delivered suicide gene therapeutics, the viral vector itself. Three outlined obstacles preventing success of anti-cancer viral vectors are as such : 1) Selective infection of cancer cells only - as CAR/other virus receptors are ubiquitous on tumour and normal tissue surfaces alike. 2) Selective replication within cancer cells only is difficult to achieve due to constitutive promoter expression of viral replicative genes. 3) Detection and elimination of viruses by immune response prevents therapeutic gene transduction. Considering the aim of lowering Ad5 dose, the mechanical viral vector delivery of a transgene to a tumour site is hindered by many obstacles such as neutralisation by pre-existing antibodies , and tissue-wide distribution of the viruses’ main receptor - CAR (coxsackie virus - adenovirus receptor). This resolves both of these issues, polymer-coating the Ad virus protects from neutralising antibodies, and also directs the virus to alternate, specific tumour surface receptors bFGF and VEGF. If we redirect the Ad5 vector to alternate docking receptors instead of CAR, we also overcome the issue that for RMS, and many cancer cell types alike, CAR receptors are in low, indistinguishable levels on cell surfaces. Regarding low CAR receptor density, one method of increasing Ad5 transfection is through overexpression of CAR receptors by stable transduction of CAR cDNA, which enhances transgene delivery for Ad-resistant cells. Gene transduction can also be enhanced by modification of the vector’s fibre knobs which bind to the receptor - either an integrin-binding RGD peptide or a polylysine peptide in the exposed HI loop were able to increase oncolysis of RMS from a conditionally replication-competent Ad vector. A conditionally replication-competent adenovirus (CRAd) demonstrates targeted infection of tumour cells, followed by replication, lysis, and release of viral progeny ; adenovirus promoters are substituted with tissue specific promoters, or alterna- tively viral replication genes are deleted and compensated for in cancer cells (which also demonstrate rapid replication). 19  To state common engineering approaches that ensure cancer-cell exclusive vi- ral replication, we must first explain adenoviral replication components and events. Early phase replication is controlled by transcripts from E1-4. E1 transcripts are subdivided into E1A and E1B. E1A transcripts produce proteins that bind or interact with p21 and CDK inhibitors , cyclin-A and E-CDK complexes , and p300/CBP transactivators to inhibit cAMP-mediated transcription. Furthermore E1A binds and sequesters P/CAF to modulate its transcriptional activity, and dis- rupt myogenesis , and can also induce p53-independent apoptosis by processing of procaspase-3. We can summarise from this that the actions of E1A transcripts are pro-apoptotic, also causing transcription of p53 by release of transcription factor E2F when E1A binds to Rb (Retinoblastoma tumour suppressor protein), and stabilisation of p53. On the other hand, E1B transcripts are anti-apoptotic as one of this transcript group’s proteins E1B - 55 kDa disables and translocates p53 to the cytoplasm , 55 kDa also aids in ubiquitination and degradation of p53 alongside the adenoviral protein E4orf6. Another E1B gene product, E1B-19kDa inhibits downstream signalling effects of p53 to prevent apoptosis, and is a functional analogue of Bcl-2 which we discussed further as having a role in GCV-mediated apoptosis. In this case E1B-19kDa acts as a Bcl-2 analogue to inhibit pro-apoptotic factors Bax/Bak from signalling for mitochondrial rupture, by inhibiting mitochondrial rup- ture, apoptosis is prevented and adenoviral replication can occur to completion. A common engineering approach to promoting apoptosis in cancer cells exclu- sively involves deletion of the E1B-19kDa and 55kDa regions. Deletion of the E1B- 19kDa gene would enable factors Bak/Bax to signal for mitochondrial rupture, cy- tochrome c release and caspase 9 activation, enabling apoptosis , therefore this strategy can be used to enhance adenoviral oncolysis, and has been demonstrated in clinical trials where E1B-19kDa deleted Ad in combination with cisplatin has a higher efficacy profile, i.e induction of apoptosis in tumorigenic tissue which succeeded a prior study excluding cisplatin, solely looking at comparative oncolytic properties of E1B-55/19 deleted Ad, ergo removal of the apoptotic-blocking gene regions. The E1B-55 kDa deletion in an adenovirus causes preferential adenoviral repli- cation in p53 dysfunctional cells which is prioritised as a method of developing oncotropism. The E1B-55 kDa region present alone in an adenovirus, with cisplatin, but no therapeutic suicide gene insert (such as Tk), has shown promise in clinical trials for advanced squamous cell carcinoma of the head and neck. 20 Figure 3.1: Comparison of control with rAAV.rh10 treated mice, for transduction ef- ficiency in skeletal muscle, measured and visualised by fluorescent imaging for EGFP intensity, and vector genome copy number quantification. 21 Chapter 4 Pro-drug Choice 4.1 Ganciclovir Ganciclovir (GCV) is normally metabolised at slow rates in mammalian cells : Viral thymidine kinase converts GCV to GCV-P, followed by conversion to GCV- TP by endogenous mammalian enzymes, GCV-TP is an inhibitor of the virus’ DNA polymerase, and competes with nucleotides for viral DNA replication. 4.2 Altering Pharmacokinetics of GCV Nucleoside analogues are widely used anti-viral agents, from numerous studies clinical success is widely stated and the mechanism of DNA replication detailed. A decade long anti-viral chemical screening process identified scopadulciol (SDC), a naturally occurring tetracyclic compound derived from the medicinal plant Scoparia Dulcis, as exhibiting strong activity against herpes virus when in combination with GCV: Largely indicative of a significant uprise in GCV-TP levels in HSV+ cells upon GCV administration, this leads us to discuss how molecular helpers, or rather stimulating substances, can be studied and applied in tandem with GCV, in the context of aiding its pharmacokinetics for the HSV-Tk/GCV therapeutic. SDC was introduced as an enhancer of ACV and inhbitior of HSV-1, via suppres- sion of viral protein synthesis in infected cells at during late-stage HSV replication ; more importantly here we are shown that SDC in combination with ACV was shown to increase quantity of dinucleotide triphosphate (dNTP) pools, alongside ACV-TP activity in the infected cells. 22 Chapter 5 Summary The aim of this report was to narrow down cellular/gene therapies for an aggressive paediatric cancer - rhabdomyosarcoma, to determine and elucidate HSV-Tk/GCV suicide gene therapy, or GDEPT. Upon a weighing and comparing the drawbacks and limitations of the specific configuration of HSV-1 Thymidine Kinase gene, Ad5 viral vector, and Ganciclovir prodrug, we consulted primary data from other reports, studies, clinical trials and journals to devise an optimisation prospectus that sought to - A) Enhance specificity (oncotropism) B) Reduce, or ideally eliminate, likeli- hood of cancer recurrence C) Increase tumour regression, and quantity of tumours regressed by the therapy. We have detailed mechanisms and components of the HSV-Tk/GCV therapeutic and propose alterations to optimise with the above criteria in mind - Therapeutic genes used - Thymidine kinase from HSV-1 serotype, with the mu- tation T62S. With SR39 and mutant 30 providing data on improved HSV1Tk - GCV kinetics. We also suggest a method (gene or promoter) for increasing Connexin 26 expression to enhance bystander effect. Promoters/enhancers with Tk - Myogen promoter, to minimise expression in off-target tissues. Cx 26 mentioned also above. Viral Vector - Adenovirus (although retroviruses are considered). Specifically we highlighted rAAV.rh10 with a high gene transduction efficiency, and Ad37 with potential for oncolytic viral therapy in rhabdomyogens. Most importantly, the E1B region was also modified, with deletion of E1B-55 and/or E1B-19 23  regions with implications on delaying/progressing apoptosis to enable/disable transcription of virus’ genes for apoptosis and release of progeny. Suicide gene therapy is a promising avenue in treatment of an aggressive cancer type in children. Although much more research data is needed to overcome risks and drawbacks, we can use several methodologies to hone in on the right one that will reassure us of clinical implementation. Word count - 6502 24 Bibliography Cancer incidence and survival among children and adolescents - pediatric monograph - seer publications 1975-1995. Ahmed Abdullah Al Awadh. 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