Dissertations / Theses on the topic 'HIV pathogenicity; Gene therapy'

Lentiviral vector delivery of anti-HIV elements could provide the basis of alternative therapies against HIV, potentially providing long term protection after a single intervention. Some primate species have evolved restriction factors formed by the fusion of TRIM5α and Cyclophilin A (TRIM5Cyp) following retrotransposition of CypA cDNA into the TRIM5 gene, which provide potent resistance against certain lentiviruses. We have designed humanised versions of these proteins combining both TRIM5 and TRIM21 with CypA, and investigated their potential for use in gene therapy against HIV-1. Both TRIM5- and TRIM21-Cyp fusion proteins provided strong restriction of HIV-1 in all of the systems tested, including primary human T cells. However, TRIM5Cyp was shown to disrupt the antiretroviral effect of endogenous TRIM5α and rescue murine retrovirus infection, whereas TRIM21Cyp caused no interference. In contrast, neither TRIM5CypA nor TRIM21CypA expression affected the antiviral activity of endogenous TRIM21. In addition to TRIMCyp restriction factors, a second anti-HIV strategy was investigated using zinc finger nucleases (ZFNs) to knockout the HIV-1 co-receptor, CCR5. ZFNs introduce a double stranded break into the CCR5 gene, which can be restored by homology directed repair. Provision of a green fluorescent protein (GFP) or TRIM21Cyp donor template exploits this repair mechanism to allow site specific integration at the CCR5 locus, although at low efficiency. Using integrating vectors, we have shown that TRIMCyp mediated restriction is so potent that no additional inhibition was conferred by CCR5 knockout. In conclusion, delivery of TRIMCyp genes using lentiviral vectors could form the basis of an intracellular vaccination strategy against HIV-1, with TRIM21Cyp having benefits by maintaining endogenous TRIM function. With further optimisation to improve efficiency, this could be combined with ZFNs for site specific integration of the transgene and knockout of CCR5 to provide a dual method of HIV-1 inhibition.

Currently, the HIV pandemic remains a major global health challenge. In order to effectively control and cure HIV-1 infection, it is necessary to perform greater research on host-HIV interactions and develop novel preventive and therapeutic approaches. The human cytidine deaminase APOBEC3G (A3G) is the first identified host restriction factor, which can serve as an initial line of defense against HIV-1 by inducing lethal mutations on proviral DNA and disrupting viral reverse transcription and integration. In order to better understand the action of A3G on HIV-1 replication, my study was focused on characterizing the interplay between A3G and HIV-1 reverse transcriptase (RT). The results indicated that A3G directly bound to RT, which contributed to A3G-mediated inhibition of viral reverse transcription. Overexpression of the RT-binding polypeptide A3G65-132 was able to disrupt wild-type A3G and RT interaction, consequently attenuating the anti-HIV effect of A3G on HIV replication. While the potent antiviral activities of A3G make it an attractive candidate for gene therapy, the actions of A3G can be counteracted by HIV-1 Vif during wild-type HIV infection. In order to overcome Vif-mediated blockage and maximize the antiviral activity of A3G, this protein was fused with a virus-targeting polypeptide (R88) derived from HIV-1 Vpr, and various mutations were then introduced into R88-A3G fusion protein. Results showed that Vif binding mutants R88-A3GD128K and R88-A3GP129A exhibited very potent antiviral activity, and blocked HIV-1 replication in a CD4+ T lymphocyte cell line as well as human primary cells. In an attempt to further determine their potential against drug resistant viruses and viruses produced from latently infected cells, R88-A3GD128K was chosen and delivered by an inducible lentiviral vector system. Expression of R88-A3GD128K in actively and latently HIV-1 infected cells was shown to be able to inhibit the replication of both drug sensitive and resistant strains of HIV-1. In conclusion, this thesis has demonstrated one of the mechanisms that how A3G can disrupt HIV-1 reverse transcription. Meanwhile, an A3G-based anti-HIV-1 strategy has been developed, which provides a proof-of-principle for a new gene therapy approach against this deadly virus.

Production of an effective vaccine and long-term treatment against human immunodeficiency virus (HIV) is elusive. In this thesis two different techniques were used in an attempt to insert HIV-neutralising monoclonal antibody (IgG1b12) sequences into a simian retroviral gene therapy agent pseudo-typed with vesicular stomatitis virus glycoprotein. Genes were encoded in either a poxvirus split-vector system or a baculovirus expression system. Both systems aim to produce replication incompetent pseudotyped virus like particles with simian origin. It is believed that the resulting non-infectious artificial lentivirus particles enter neighbouring cells, penetrate the nucleus and insert genetic material (the antibody gene) into the mammalian genome. The poxvirus split-vector system used in this project was a Vaccinia Retroviral Hybrid Vector, where recombinant modified vaccinia Ankara (MVA) is used to deliver the simian immunodeficiency virus (SIV) like particles into mammalian cells. However, the MVA system failed to express proteins of interest due to the instability of genetic insertion into the recombinant MVA genome. As an alternative strategy, two different BacMam systems were used to allow the production of VLPs, where mammalian cells are co-transduced with different recombinant baculoviruses (rBVs). VLPs were expressed either under the control of T7 RNA polymerase system or under the cytomegalovirus immediate early gene promoter. The results from the first BacMam system indicated that the T7 RNA polymerase system was not suitable to express detectable levels of proteins. The results indicated that translation of the produced mRNA by T7 promoter is inefficient, most likely because of the absence of RNA 5’ cap structure. To overcome this hybrid BV–T7 system limitation, a different system was developed. Proteins of interest from the second BacMam system were successfully expressed and detected using western blot analysis. VLPs were generated and visualised under electronic microscope. IgG1b12 was secreted in the supernatant of the transduced mammalian cells. Mammalian cells were successfully transduced with multiple different recombinant BVs simultaneously. The study establishes the feasibility of antibody gene transfer, and demonstrates the use of SIV like particles production to transduce mammalian cells using BacMam technology. The technique may have application for use as an immunotherapy of HIV infection as well providing long-acting prevention of HIV infection for those not yet infected with HIV.

Microbiology and Immunology
Ph.D.
Since it emerged as an infectious agent in 1981, the human immunodeficiency virus type 1 (HIV-1) is continually disseminated and remain fatal to the majority of those infected. Strategies including highly active retroviral therapies (HAART) with nucleoside analogues and protease inhibitors have shown limited success in therapy due to the virus' ability to evolve rapidly at every replication cycle as a consequence of it's highly error prone reverse transcriptase, generating resistant retroviral strains and in addition to latent HIV-1 reservoirs. Thirty years of research efforts to find a cure or to generate a vaccine has been met with failure. It is, therefore, of necessity to broaden our paradigm of therapy for the treatment and eventual cure of HIV-1 infection. In this study, I look beyond the current anti-retroviral strategies and instead rely on the mammalian host immune system to inhibit HIV-1 replication through molecular genetic manipulation. Here, we approach the inhibition of HIV-1 replication by up-regulation of the innate antiviral pathway that is natural to mammalian cells. HIV-1 derived self-inactivating lentiviral (SIN) vectors were designed and constructed to deliver the antiviral payloads of two antiviral enzymes, p68 kinase (PKR) and 2'-5' oligoadenlyate synthetase (2-5OAS), to target cell, SupT1 lymphoblastoid cells and CD4+ T lymphocytes under the control of a constitutive cytomegalovirus (CMV) promoter. These data here demonstrates a significant inhibition of HIV-1 replication in cells transduced with the anti HIV-1 transgenes PKR and 2-5OAS as determined by HIV-1 induced syncytia formation and HIV-1 p24 antigen capture assay. Furthermore, here demonstrated is an increase up-regulation of PKR and 2-5OAS 96 hr post cell transduction in all the clones when compared to pHIV empty vector control. These results demonstrate that the over-expression of PKR and 2-5OAS can inhibit HIV-1 replication and also confirm the involvement of PKR and 2-5OAS in the IFN-associated antiviral pathway against HIV-1 infection.
Temple University--Theses

Gene therapy is the introduction or alteration of genetic material with the intention to treat disease. To support this aim, viruses have been modified, with elements linked to viral pathogenicity removed from their genome and replaced by the genetic material to be delivered. Gene therapy vectors based on lentiviruses have many advantages, such as the ability to transduce non-dividing cells and to target specific cell types via pseudotyping. They have been successfully used in ex vivo clinical trials for several haematopoietic stem cell disorders. Lentiviral vectors, however, suffer from substantially lower titres than the more popular adeno-associated virus (AAV)-based vectors and therefore have limited applicability for in vivo gene therapy which requires much greater quantities of virus. The main aim of this thesis was to investigate strategies to improve lentiviral vector productivity during manufacture, in order to increase the likelihood of lentiviruses being adopted for disease treatment. Initial experiments were based on the lentiviral vector manufacturing process currently being developed by the United Kingdom Cystic Fibrosis Gene Therapy Consortium for the generation of highly concentrated, purified lentivirus for clinical use. Supplementation of FreeStyle 293 Expression Medium used during upstream processing was attempted, but none of the assessed supplements led to significant increases in lentiviral vector production. Investigation into intrinsic immunity to viral infection indicated that over-expression of the protein kinase RNA-activated (PKR) led to lower production titres, but over-expression of its inhibitors was not successful at increasing titres. The focus then shifted to reducing, or 'knocking-down', inhibitory factors present in the host cells, which could adversely affect viral titres. Investigation of the published HIV-1 literature revealed a possible 152 candidate inhibitory factors described as having a negative impact on HIV-1 replication in the late stages of the life cycle of the virus. A novel siRNA screen was developed to assess the effect of ‘knock-down' of inhibitory factors on lentiviral vector titre. Application of the screen to 89 candidate inhibitory factors identified nine genes which, when knocked-down, resulted in increased lentiviral vector production by more than 40%. Further work will be necessary to understand the role of the inhibitory factors in lentiviral vector production, but novel cell lines in which genes encoding these factors have been permanently deleted from producer cells could lead to higher titres, reducing costs in the manufacture of lentiviral vectors and making in vivo gene therapy more feasible from a health economics perspective.

South Africa is in dire need of a public umbilical cord blood stem cell bank (UCB SCB). A severe shortage of genetically compatible samples for BM transplantation precludes the majority of South Africans from receiving the relevant medical care. UCB is a viable alternative to BM but is currently disposed of post-delivery. UCB could furthermore serve as a resource of genetically compatible haematopoietic progenitor cells (HPCs) that could be used in gene therapy approaches directed towards a cure for HIV-1. Knowing whether HIV-1 affects or infects primitive HPCs is vital to determine the course of action for transplantation of UCB-derived genetically resistant HPCs. Collecting and storing UCB in a public UCB bank could thus serve as a vital resource of genetically compatible samples for BM transplantation. It was thought that the high incidence of HIV-1 in South African patients and the persistent stigma surrounding HIV-1 would be problematic for collecting sustainable numbers of UCB units and subjecting units to compulsory screening for infectious diseases. This was however, not the case. In the South African context, we are faced with unique and rich challenges relating to cultural and religious differences that are further augmented by linguistic constraints and educational insufficiencies. Nevertheless, the majority of patients within the interviewed patient cohort were supportive of the idea of establishing a public UCB SCB in SA and were willing to undergo additional HIV-1 screening. The Ultrio-Plus® assay was verified in this study for screening UCB units for HIV-1 and could be used in routine analyses of UCB units prior to banking. Conflicting results in the literature exist with regard to HIV-1’s ability to infect or affect haematopoietic progenitor cells. Results from this study revealed that HIV-1 was not only able to affect HPCs’ ability to form colonies in vitro, but was also capable of infecting CD34+ HPCs in some individuals. These results substantiate the theory that some CD34+ HPCs serve as viral reservoirs which could account for residual viraemia in patients on antiretroviral therapy. Results suggest that allogeneic transplantation of HIV-1 infected individuals with UCB-derived, genetically modified HPCs, should be pursued.
Thesis (PhD)--University of Pretoria, 2012.
Immunology
unrestricted

Thesis (MSc)--Stellenbosch University, 2012.
Background: While Selection of reverse transcriptase (RT) mutation has been reported frequently, protease (PR) mutations on antiretroviral therapy (ART) including boosted Protease inhibitor (PI) have not been reported as much in Zambia. Affordable in-house genotyping assays can been used to expand the number of patients receiving drug resistance geno-typing, which can aid in determining prevalence of RT/PI emerging mutations. Methods: A previously published drug resistance genotyping assay was modified and used to genotype RT and PR genes. 19 patients virologically failing first-line regimen and 24 failing second-line regimen were studied to determine resistance patterns. Virological failure was defined as failing to maintain <1000 copies/mL during ART. Only major and minor RT and PR mutations (IAS-USA 2010) were considered for analysis. The in-house assay was validated by comparing sequence data of 7 previously ViroSeq tested samples and 5 randomly selected samples to determine reproducibility. Results: The in-house assay efficiently amplified all 12 validation samples with the lowest sample scoring 99.4% sequence homology. The most common RT mutation was M184V (79% n=19) and (71% n=24) first and second-line respectively. No significant differences were reported in all the other RT mutations between first-line and secondline regimens. Drug resistant PI mutations (I54V, M46I and V82A all present 20.8%) were only found in the second-line regimen and were insignificant, p= 0.0562. Conclusion: The in-house assays can be used as alternatives for commercial kits to genotype HIV-1C in Zambia without compromising test quality. The insignificant PI drug resistant mutations which were found, despite virological failure in patients, could indicate a possibility of other mutations within the HIV-1 genome that could reduce PI susceptibility.

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CAPES
CNPq
HIV/aids (Vírus da Imunodeficiência Humana/aids) é considerado uma pandemia, envolvendo mais de 70 milhões de infecções e 35 milhões de mortes desde o primeiro relato na década de 80. O HIV tipo 1 (HIV-1) infecta principalmente linfócitos T CD4+ e linhagens de macrófagos, tendo sua patogenicidade definida pela depleção de LT CD4+. Além disso, a condição de infecção por HIV-1 é bastante complexa e dependente de diversos fatores relacionados à variabilidade dos indivíduos no que diz respeito à suscetibilidade à infecção e à progressão para a aids, sendo observada a ativação imunológica generalizada. Envolvida na modulação das respostas imunes inata e adaptativa encontra-se a vitamina D, que desempenha papel no metabolismo mineral e apresenta efeito pleiotrópico no crescimento e diferenciação celulares. Seus efeitos imunológicos são dados a partir da ligação com o receptor de vitamina D (VDR) de diversas células, regulando a liberação de citocinas, a função e proliferação de linfócitos T e a produção de peptídeos antimicrobianos como a catelicidina. O VDR atua modulando a ação dessa vitamina induzindo a resposta imune local e variações genéticas presentes no gene codificador do VDR podem levar à diminuição de sua atividade e, consequentemente, ao prejuízo para o papel da vitamina D. Nos indivíduos infectados pelo HIV, os níveis de deficiência dessa vitamina são altos e fatores como raça, insuficiência renal, pouca exposição à luz ultravioleta e exposição as drogas anti-HIV, como o Efavirenz, estão associados a essa deficiência, respectivamente, sendo determinantes para a susceptibilidade à infecção pelo HIV e a predição da progressão da doença. Sendo assim, neste trabalho foram estudados seis polimorfismos de base única (SNPs) (rs3890733, rs476048, rs1540339, rs2248098, rs2228570 e rs11568820) presentes no gene do receptor de vitamina D (VDR) e sua influência na resposta dos pacientes à Terapia Antirretroviral (TARV). Foram recrutados 107 pacientes acompanhados e tratados no Hospital Dia do Instituto de Medicina Integral Professor Fernando Figueira (IMIP), subdivididos em quatro grupos: I- Sucesso Terapêutico, II- Falha Terapêutica, III- Sucesso Imunológico, IV- Falha Imunológica, e analisadas variáveis clínicas e epidemiológicas, como gênero, idade, peso e etnia. Não foram observadas associações estatísticas nas análises isoladas entre os polimorfismos dos genes do VDR com a falha virológica ou a resposta imunológica. Porém, nas análises multivariadas, o genótipo C/C do rs1540339 mostrou-se associado com o gênero no sucesso virológico (OR=0,08, p=0,04). Em adição, a análise envolvendo peso, etnia e gênero e o rs3890733 mostrou associação com a resposta imunológica para os genótipos C/C e T/T no modelo sobredominante (OR=0,21, p=0,024). Os resultados indicam a importância do receptor de vitamina D em infecções por HIV-1 e poderão contribuir para o entendimento da variabilidade das respostas dos pacientes à TARV.
HIV/aids (Human Immunodeficiency Virus/aids) is considered a pandemic, involving more than 70 million infections and 35 million deaths since the first report in the 80’s. HIV type 1 (HIV-1) infects mainly T lymphocytes CD4 + and macrophage lineages, and their pathogenicity is defined by the depletion of CD4 +. Furthermore, the condition of HIV- 1 infection is very complex and dependent on many factors related to the individual variability, regarding the susceptibility to infection and progression to AIDS, generalized immune activation being observed. Involved in the modulation of innate and adaptive immune responses is vitamin D, which plays a role in mineral metabolism and has pleiotropic effects on cell growth and differentiation. Their immune effects are data from binding to the vitamin D receptor (VDR) in various cells, regulating the release of cytokines, the function and proliferation of T lymphocytes and the production of antimicrobial peptides as cathelicidin. The VDR acts modulating the action of vitamin D by inducing local immune responses and genetic variations present in the VDR encoding gene can lead to reduction of its activity and consequently, disfunction in the role of vitamin D. In HIV-infected individuals, this vitamin deficiency levels are high and factors such as race, kidney failure, lower exposure to ultraviolet light and exposure to anti- HIV drugs, such as Efavirenz, are associated with this deficiency, being determinants on the susceptibility to HIV infection as well as prediction of disease progression. Therefore, in this work we studied six single nucleotide polymorphisms (SNPs) (rs3890733, rs476048, rs1540339, rs2248098, rs2228570 and rs11568820) present in the D vitamin receptor gene (VDR) and its influence on patients’ response to Antiretroviral Therapy (ART). We recruited 107 patients followed from the Hospital Day Integrative Medicine Institute Professor Fernando Figueira (IMIP), subdivided into four groups: I. Therapeutic Success, II. Therapeutic Failure, III. Immune Success, IV. Immune Failure, and analyzed clinical and epidemiological variables, such as gender, age, weight and ethnicity. No statistically significant associations were observed in the isolated analyzes between polymorphisms of the VDR gene with therapeutic failure or immune response. However, in multivariate analyzes, the rs1540339 C/C genotype was associated with gender in therapeutic success (OR = 0.08, p = 0.04). In addition, analysis involving weight, ethnicity and gender and the rs3890733 showed association with the immune response to the C/C genotype and T/T in overdominant model (OR = 0.21, p = 0.024). The results indicate the importance of vitamin D receptor in HIV- 1 infections and may contribute to the understanding of variability of patient’s various responses to ART.

Despite 30 years of intensive research，an effective human immunodeficiency virus (HIV) vaccine still remains elusive. The desirable immune response capable of providing protection against HIV acquisition is still not clear. The accumulating evidence learned from a recent vaccine efficacy correlate study not only confirmed the importance of antibody responses, but also highlighted potential protective functions of antibodies with a broad repertoire of HIV-1 epitope specificities and a wide range of different antiviral mechanisms. This necessitates a deep understanding of the complexity and diversity of antibody responses elicited by HIV-1 vaccines. My dissertation characterizes antibody response profiles of HIV-1 Env antibodies elicited by several novel immunogens or different immunization regimens, in terms of magnitude, persistence, epitope specificity, binding affinity, and biological function. First, to overcome the challenge of studying polyclonal sera without established assays, we expanded a novel platform to isolate Env-specific Rabbit mAbs (RmAb) elicited by DNA prime-protein boost immunization. These RmAbs revealed diverse epitope specificity and cross-reactivity against multiple gp120 antigens from more than one subtype, and several had potent and broad neutralizing activities against sensitive Tier 1 viruses. Further, structural analysis of two V3 mAbs demonstrated that a slight shift of the V3 epitope might have a dramatic impact on their neutralization activity. All of these observations provide a useful tool to study the induction of a desired type of antibody by different immunogens or different immunization regimens. Since heavily glycosylated HIV Env protein is a critical component of an HIV vaccine, we wanted to determine the impact of the HIV Env-associated glycan shield on antibody responses. We were able to produce Env proteins with a selective and homogeneous pattern of N-glycosylation using a glycoengineered yeast cell line. Antigenicity of these novel Env proteins was examined by well-characterized human mAbs. Immunogenicity studies showed that they were immunogenic and elicited gp120- specific antibody responses. More significantly, sera elicited by glycan-modified gp120 protein immunogens revealed better neutralizing activities and increased diversity of epitopes compared to sera elicited by traditional gp120 produced in Chinese Hamster Ovary (CHO) cells. Further, we examined the impact of the delivery order of DNA and protein immunization on antibody responses. We found that DNA prime-protein boost induced a comparable level of Env-specific binding Abs at the peak immunogenicity point to codelivery of DNA. However, antibody responses from DNA prime-protein boost had high avidity and diverse specificities, which improved potency and breadth of neutralizing Abs against Tier 1 viruses. Our data indicate that DNA vaccine priming of the immune system is essential for generation of high-quality antibodies. Additionally, we determined the relative immunogenicity of gp120 and gp160 Env in the context of DNA prime-protein boost vaccination to induce high-quality antibody responses. Immunized sera from gp120 DNA primed animals, but not those primed with gp160 DNA, presented with distinct antibody repertoire specificities, a high magnitude of CD4 binding site-directed binding capabilities as well as neutralizing activities. We confirmed the importance of using the gp120 Env form at the DNA priming phase, which directly determined the quality of antibody response.

Despite 30 years of intensive research，an effective human immunodeficiency virus (HIV) vaccine still remains elusive. The desirable immune response capable of providing protection against HIV acquisition is still not clear. The accumulating evidence learned from a recent vaccine efficacy correlate study not only confirmed the importance of antibody responses, but also highlighted potential protective functions of antibodies with a broad repertoire of HIV-1 epitope specificities and a wide range of different antiviral mechanisms. This necessitates a deep understanding of the complexity and diversity of antibody responses elicited by HIV-1 vaccines. My dissertation characterizes antibody response profiles of HIV-1 Env antibodies elicited by several novel immunogens or different immunization regimens, in terms of magnitude, persistence, epitope specificity, binding affinity, and biological function. First, to overcome the challenge of studying polyclonal sera without established assays, we expanded a novel platform to isolate Env-specific Rabbit mAbs (RmAb) elicited by DNA prime-protein boost immunization. These RmAbs revealed diverse epitope specificity and cross-reactivity against multiple gp120 antigens from more than one subtype, and several had potent and broad neutralizing activities against sensitive Tier 1 viruses. Further, structural analysis of two V3 mAbs demonstrated that a slight shift of the V3 epitope might have a dramatic impact on their neutralization activity. All of these observations provide a useful tool to study the induction of a desired type of antibody by different immunogens or different immunization regimens. Since heavily glycosylated HIV Env protein is a critical component of an HIV vaccine, we wanted to determine the impact of the HIV Env-associated glycan shield on antibody responses. We were able to produce Env proteins with a selective and homogeneous pattern of N-glycosylation using a glycoengineered yeast cell line. Antigenicity of these novel Env proteins was examined by well-characterized human mAbs. Immunogenicity studies showed that they were immunogenic and elicited gp120- specific antibody responses. More significantly, sera elicited by glycan-modified gp120 protein immunogens revealed better neutralizing activities and increased diversity of epitopes compared to sera elicited by traditional gp120 produced in Chinese Hamster Ovary (CHO) cells. Further, we examined the impact of the delivery order of DNA and protein immunization on antibody responses. We found that DNA prime-protein boost induced a comparable level of Env-specific binding Abs at the peak immunogenicity point to codelivery of DNA. However, antibody responses from DNA prime-protein boost had high avidity and diverse specificities, which improved potency and breadth of neutralizing Abs against Tier 1 viruses. Our data indicate that DNA vaccine priming of the immune system is essential for generation of high-quality antibodies. Additionally, we determined the relative immunogenicity of gp120 and gp160 Env in the context of DNA prime-protein boost vaccination to induce high-quality antibody responses. Immunized sera from gp120 DNA primed animals, but not those primed with gp160 DNA, presented with distinct antibody repertoire specificities, a high magnitude of CD4 binding site-directed binding capabilities as well as neutralizing activities. We confirmed the importance of using the gp120 Env form at the DNA priming phase, which directly determined the quality of antibody response.

Human immunodeficiency type 1 (HIV-1) is able to elicit broadly potent neutralizing antibodies in a very small subset of individuals only after several years’ infection and as a result, vaccines that elicit these types of antibodies have been difficult to design. The RV144 trial showed that a moderate protection is possible, which may correlate with antibody dependent cellular cytotoxicity (ADCC) activity. Previous studies in the Lu lab demonstrated that in an HIV-1 vaccine phase I trial, DP6-001, a polyvalent Env DNA prime-protein boost formulation, could elicit potent and broadly reactive, gp120-specific antibodies with positive neutralization activities along with multiple Fc mediated effector functions. I developed a protocol for the production and analysis of HIV-1 Env-specific human monoclonal antibodies (mAbs) isolated from these DP6-001 vaccinees. By utilizing a labeled gp120 bait to isolate Env specific B cells, paired heavy and light chain immunoglobulin (Ig) genes were cloned and allowed for the production of monoclonal antibodies with specificity for gp120. By using this protocol, 13 isolated mAbs from four DP6-001 vaccinees showed broad binding activities to gp120 proteins of diverse subtypes, both autologous and heterologous to vaccine immunogens, with mostly conformational epitopes and a few V3 and C5 specific mAbs. Equally cross-reactive Fc-mediated functional activities, including ADCC and antibody dependent cellular phagocytosis (ADCP), were present with both immune sera and isolated mAbs, confirming the induction of non-neutralizing functional antibodies by the DNA prime- protein boost vaccination. Elicitation of broadly reactive mAbs by vaccination in healthy human volunteers confirms the value of the polyvalent formulation in this HIV-1 vaccine design.

Human immunodeficiency type 1 (HIV-1) is able to elicit broadly potent neutralizing antibodies in a very small subset of individuals only after several years’ infection and as a result, vaccines that elicit these types of antibodies have been difficult to design. The RV144 trial showed that a moderate protection is possible, which may correlate with antibody dependent cellular cytotoxicity (ADCC) activity. Previous studies in the Lu lab demonstrated that in an HIV-1 vaccine phase I trial, DP6-001, a polyvalent Env DNA prime-protein boost formulation, could elicit potent and broadly reactive, gp120-specific antibodies with positive neutralization activities along with multiple Fc mediated effector functions. I developed a protocol for the production and analysis of HIV-1 Env-specific human monoclonal antibodies (mAbs) isolated from these DP6-001 vaccinees. By utilizing a labeled gp120 bait to isolate Env specific B cells, paired heavy and light chain immunoglobulin (Ig) genes were cloned and allowed for the production of monoclonal antibodies with specificity for gp120. By using this protocol, 13 isolated mAbs from four DP6-001 vaccinees showed broad binding activities to gp120 proteins of diverse subtypes, both autologous and heterologous to vaccine immunogens, with mostly conformational epitopes and a few V3 and C5 specific mAbs. Equally cross-reactive Fc-mediated functional activities, including ADCC and antibody dependent cellular phagocytosis (ADCP), were present with both immune sera and isolated mAbs, confirming the induction of non-neutralizing functional antibodies by the DNA prime- protein boost vaccination. Elicitation of broadly reactive mAbs by vaccination in healthy human volunteers confirms the value of the polyvalent formulation in this HIV-1 vaccine design.

Le traitement du cancer par thérapie génique nécessite d’une part des gènes suicides efficaces et, d’autre part, l’adressage spécifique de ces gènes aux cellules cancéreuses. J'ai d'abord caractérisé un nouveau gène suicide dérivé de la désoxycytidine kinase humaine (dCK) : le M36. Comparé à la dCK, le M36 permet une meilleure sensibilisation des certaines cellules cancéreuses aux traitements avec différents chimiothérapeutiques comme la gemcitabine et la cytarabine. Ces résultats sont particulièrement encourageants pour l'élimination des cellules cancéreuses résistantes à ces traitements du fait d’un défaut de la dCK. Dans une deuxième partie, je me suis intéressée à l'adressage spécifique des transgènes aux cellules cancéreuses par les vecteurs lentiviraux. J'ai travaillé à la preuve de concept qu’une enveloppe (Env) VIH modifiée peut permettre un tel ciblage. J'ai généré une Env qui a fortement diminué son tropisme naturel et qui comporte un motif liant le marqueur tumoral modèle HER2
Cancer gene therapy requires the use of an effective suicide gene and the specific targeting of cancer cells. In my PhD work, I have first characterized a new potential suicide gene derived from human deoxycytidine kinase (dCK): M36. Compared to dCK, M36 improves sensitization of certain cancer cells to treatment with chemotherapeutic compounds as gemcitabine and AraC. These results are particularly encouraging for the elimination of cancer cells resistant to the treatment because of a defect with dCK. In a second part, I have worked at the proof of concept that a modified HIV envelope can allow specific targeting of cancer cells by lentiviral vectors. During this work, I have generated a CD4i envelope with a strongly diminished natural tropism and that carries a motif known to bind the model cell surface cancer marker HER2. This envelope constitutes a good starting material to be improved by evolution in cell culture to obtain specific targeting of HER2+ cells

Tese de doutoramento em Farmácia (Microbiologia), apresentada à Universidade de Lisboa através da Faculdade de Farmácia, 2008
The spread of HIV-1 has been dramatic since the early eighties, when the virus was discovered as the causative agent of AIDS. In the absence of an effective vaccine against HIV, a worldwide search has been made in the past two decades to develop small-molecule inhibitors to target essential steps in the viral cycle. Over the recent years, gene therapy has been highly regarded as a new form of molecular medicine in treatment of HIV/AIDS, either asan alternative or as a complement to anti-retroviral chemotherapy. An intrabody consists of an antibody designed to be expressed intracellularly and directed to different subcellular compartments where they can exert their function more effectively. The binding of an intrabody to its molecular target has the potential to block, suppress, alter or even enhance the process mediated by that molecule. Within this context, intracellular antibodies (intrabodies) represent a new class of neutralizing molecules with potential use in gene therapy approaches. The HIV 1 integrase (IN) protein is currently the focus of an intense research effort to develop new anti-HIV-1 drugs. This enzyme catalyses the integration of HIV genome into the chromosome of the host cell, arguably the most insidious step in the infection process. In the first project of this thesis (Chapter 2), we explored the intracellular immunization approach by developing rabbit intrabodies against the HIV-1 IN protein. We immunized rabbits with HIV-1 IN and developed a combinatorial scFv library againstIN. We were able to identify 5 different scFv's antibodies with high binding activity and specificity to IN. These scFv's bound simultaneously to the catalytic and C-terminus domains of IN. In addition, these antibodies have the ability to inhibit the strand transfer processing. Intrabody-expressing cells, either in their cytoplasm or nuclear compartments, were highly resistant to HIV-1 infection. Importantly, when HIV-1 particles where produced in the presence of anti-IN scFv, the expression of intrabodies did not affect virion production significantly. However, it markedly reduced the infectivity of progeny virions due to the incorporation of anti-IN scFv into the viral particles. These findings provide proof-in-principle that rabbit anti- IN intrabodies can be designed to block early and late stages of HIV-1 replication. As a result, our intrabodies might be useful agents for "intracellular immunization"- and used as new tools to study the structure and function of HIV-1 IN due to their epitope binding characteristics. Another potential target for HIV-1 treatment is Vif. This viral protein overcomes the innate antiviral activity of a cytidine deaminase APOBEC3G that induces G to A hypermutation in the viral genome, resulting in enhancement of viral replication infectivity. We previously demonstrated that anti-Vif scFv and camelized VH intrabodies are an effective approach to inhibit this crucial step of the viral replication cycle. In the second project of this thesis (Chapter 3), we showed that the rabbit VL domain can also be very potentially used as an intrabody. Our results demonstrate that the anti- Vif VL single-domain preserve the antigen-binding activity and specificity in the absence of the parent VH domain. In addition, the VL single domain was highly expressed in microbial cell culture and show favourable biophysical properties. The expression of the VL intrabody in eukaryotic cells also showed that the rabbit VL was correctly folded as soluble protein in the reducing environment and could strongly neutralize HIV-1 infectivity. Therefore, the present study suggests that rabbit VL single-domains have also an enormous value as intracellular antigen recognition units. Lentiviral vectors are among the most efficient tools for gene delivery into mammalian cells. A major goal of lentiviral gene delivery systems is to develop vectors that can efficiently target specific cell types. In the last project of this thesis (Chapter 4), we attempted to generate viral particles for targeting gene delivery. To achieve this goal we have used CCR5-positive cells as the target for our strategy. We designed a novel Sindbis pseudotyped vector where the Sindbis E2 receptor binding envelope protein was modified to directly encode a scFv against the CCR5 chemokine receptor. Targeting into specific cells was mediated by the anti-CCR5 scFv display, and viral titers were close to 106 EGFP transduction units/ml. Our data demonstrate that the length of the peptide linker that connects the heavy chain and light chain of anti-CCR5 scFv significantly affects the efficiency of infection. Infection levels obtained with Sindbis envelope displaying a scFv with a longer linker was consistently higher than that with Sindbis envelope displaying a scFv with a short linker. The results presented show that chimeric scFv-Sindbis pseudotyped lentiviral vectors have the potential to become an efficient and broadly applicable approach for targeting gene delivery to specific cells. Furthermore, this strategy has the potential to become a powerful approach for targeting gene delivery in anti- HIV gene therapy due to the important role of CCR5 expression in disease progression.
Fundação para a Ciência e a Tecnologia (FCT), (SFH/BD/17039/2004)

Indiana University-Purdue University Indianapolis (IUPUI)
Lentiviral vector mediated gene therapy has made great strides in recent years with several successful clinical trials. However, adverse events encountered with some early trials have highlighted the necessity to improve upon its safety. Improvements can range from early steps in vector production to evaluation of insertion sites post-transduction. We have evaluated an FDA approved DNase for removal of residual plasmid DNA during vector production, developed novel non-integrating lentiviral vectors and employed modified insertion site analysis post-transduction to improve the safety of lentiviral vector mediated gene therapy. To prevent the exposure of gene therapy patients to HIV-1 DNA it is essential to remove residual plasmid DNA during vector production. We evaluated a recombinant human DNase which has been FDA approved for use in patients as an alternative to a bacterially derived DNase. Our results indicate this DNase is an effective alternative with a potentially safer profile for use in patients. The ability of lentiviral vectors to stably integrate their genome into a host cell’s DNA can have negative side-effects due to the risk of insertional mutagenesis. Non-integrating lentiviral vectors have been developed to alleviate this risk in applications where integration is not necessary. However, a low frequency of illegitimate integration persists when using these vectors. We have developed a novel non-integrating vector mutation and evaluated the efficacy of combining it with other mutations for reducing the frequency of illegitimate integration. We demonstrate that combining mutations that inhibit integration can further reduce the frequency of illegitimate integration. Several methodologies have been developed for evaluating the insertion sites of normal integrating lentiviral vectors. Illegitimate integration by non-integrating vectors demonstrates mechanisms which result in insertions and/or deletions at the vector-genome junction. Current methods lack the sensitivity to account for these variables in a high-throughput manner. We have adapted modifications to current methods to improve the capture of these variable insertion sites for analysis. The results of these studies improve the safety of lentiviral vector mediated gene therapy by improving the purity of the vector product, providing a safer vector for non-integrase mediated applications, and allowing more sensitive analysis of insertion sites post-transduction.

AIDS is caused by a lentivirus, HIV-1. In addition to antiretroviral drugs that are currently in use for HIV/AIDS therapy, a number of gene therapy strategies have been designed as alternative therapies. Most of these therapies target HIV RNA/proteins, which are subject to high rate of mutation, resulting in escape mutants. Viral entry is mediated by CCR5 co-receptor in most routes of transmission. To downregulate CCR5 as a gene therapy approach, we targeted seven unique sites within the CCR5 mRNA by a multimeric hammerhead ribozyme, Rz1-7. Hammerhead ribozyme is a small RNA that cleaves a target RNA upon binding to it. Expressing the Rz1-7 from HIV-1- and MSCV-based vectors in otherwise susceptible cells inhibited replication of a CCR5-tropic strain of HIV-1 by 99-100%. The Rz1-7 will be tested for inhibition of HIV-1 replication in the CD4+ T-lymphoid and myeloid progeny of transduced human CD34+ hematopoietic progenitor stem cells. It may be preferable to interfere HIV-1 life cycle at the DNA level since a one-time inactivation might suffice to confer a complete and permanent inhibition of virus replication in the gene modified cells and their progeny. This is what other strategies that target the HIV-1 RNA/protein can hardly offer. For this purpose, group II introns, which are able to splice out and get incorporated into a specific DNA sequence, can be designed/modified to gain novel DNA targeting specificities. As a novel approach, we have examined whether insertion of a modified intron into an infectious HIV-1 clone at two sites within the integrase domain of HIV-1 pol gene could inhibit virus replication. Intron insertion into the HIV-1 clone was induced and mammalian cells were transfected with intron-inserted HIV-1 clones. Although similar amounts of HIV-1 RNA, protein, and progeny virus were produced from the clones as from wild-type HIV-1 provirus DNA, in the absence of a functional integrase, the HIV-1 reverse-transcribed DNA failed to integrate and virus replication was aborted. These results demonstrate that modified group II introns can confer complete inhibition of virus replication at the level of second round of infection. We are now developing vectors to assess whether intron insertion can take place in mammalian cells.

O uso de vetores lentivirais baseados no Vírus da imunodeficiência humana em ensaios clínicos de terapia génica tem vindo a aumentar devido à sua capacidade de transduzir e modificar permanentemente tanto células não quiescentes como quiescentes, e ao seu padrão de integração considerado mais seguro do que o dos vetores retrovirais. Para tirar partido de todo o potencial destes vetores em terapia génica, são desejáveis linhas celulares produtoras estáveis. Porém, o seu desenvolvimento tem sido dificultado pela citotoxicidade de alguns componentes virais, nomeadamente a protease. Neste trabalho de Mestrado, uma protease geneticamente modificada e menos tóxica foi avaliada para produção de vetores lentivirais. Embora menos citotóxica, esta protease é menos ativa. Enquanto para glicoproteínas do invólucro que não requerem processamento proteolítico – VSV-G – a menor atividade da protease não afetou o título do vetor, a conjugação desta protease com glicoproteínas utilizadas tipicamente no desenvolvimento de células produtoras estáveis – 4070A, GaLV10A1 e RD114A – resultou num título menor. Assim, foi alterado o local de clivagem da protease nestas glicoproteínas por meio de engenharia genética. A modificação dos locais de clivagem da protease no GaLV10A1 permitiu recuperar os títulos virais para níveis semelhantes aos obtidos com a protease wild-type, em produção transiente, com um aumento de 5,5 a 36,7 vezes. As glicoproteínas modificadas estão, de momento, a ser implementadas em linhas celulares produtoras estáveis, que expressam a protease modificada. Este trabalho contribui para o desenvolvimento de uma nova linha celular “empacotadora” estável para a produção contínua de vetores lentivirais, descrevendo primeiramente o uso da protease modificada e fornecendo novas glicoproteínas quiméricas, nomeadamente GaLV10A1giflet. Além disso, esta tese abriu a porta para o uso de uma nova glicoproteína modificada com maior título – GaLV10A1ΔR – para o desenvolvimento de linhas celulares produtoras estáveis recorrendo à estratégia de knock out do recetor celular de GaLV10A1.

CCR5 is the major co-receptor for HIV-1 entry and an important target for drug development. The recent finding that hematopoietic stem cell [1] transplantation from a CCR5-negative donor to an HIV-infected patient produces long-term virus control in the absence of antiretroviral drugs suggests the potential of stem cell and gene therapies targeting CCR5. To this end, we are developing a protocol to knock-out CCR5 in a patient's own HSC using CCR5-targeted zinc finger nucleases (ZFNs) ZFNs are sequence specific proteins that generate a double-stranded break in DNA, which is converted into a gene-disrupting lesion by host repair processes. We have optimized the delivery and function of ZFNs targeting CCR5 in human CD34-positive HSC, achieving up to 27% disruption of CCR5 alleles. We confirmed that ZFN-treated HSC remain fully functional by transplanting a mouse model of human hematopoiesis, the NOD/SCID/IL2gammacnull (NSG) mouse, where the modified HSC retained the ability to differentiate into multiple hematopoietic lineages Humanized NSG mice are additionally capable of supporting HIV-1 infection. Following challenge with an R5-tropic virus, control animals demonstrated altered CD4:CD8 ratios, profound loss of human cells in the thymus and GALT, and high viral loads in multiple tissues sampled. In contrast, ZFN-treated animals had significantly lower acute viral loads and very low levels of virus in tissues by 10-12 weeks post-infection. At this stage the numbers of human cells in tissues that are targets for HIV-1 infection had normalized, including the GALT and thymus. FACS and PCR analysis revealed a rapid and dramatic selection for CCR5-negative cells in these populations. These findings demonstrate that ZFN-treated HSC can generate HIV-resistant cells in vivo that rapidly replace cells depleted by HIV-1 infection, and importantly, preserve GALT populations. Transient ZFN treatment resulting in permanent disruption of CCR5 in autologous HSC could therefore represent a viable clinical approach for HIV-infected patients
acase@tulane.edu

Vectors derived from Human Immunodeficiency Virus type 1 (HIV-1) are being widely developed for gene therapy applications, principally because they are able to transduce both dividing and non-dividing cells and result in stable, long term gene expression. However, these vectors are difficult to produce in high titres and sufficient volumes for large scale experiments and clinical application. Therefore, an investigation into methods to improve the production of HIV-1 derived gene transfer vectors was undertaken. One factor that limits the production of recombinant virus is the amount of viral genomic RNA available for packaging into virions. Therefore, a transfer vector was modified with the aim of increasing the amount of genomic RNA produced. Substitution of the polyadenylation (pA) signal, mutation splice donor sites and removal of unnecessary sequences were all examined. pA signal readthrough was quantified to determine the effect of these modifications on the rate of pA signal readthrough. Insertional mutagenesis and vector mobilisation are recognised risk factors with all integrating vectors. Self inactivating (SIN) vectors, which contain a deletion of U3 sequences in the 3’ LTR, demonstrate a reduced rate of mobilisation. Transduction with these vectors results in a provirus containing no viral promoter elements, with transcription of the transgene being controlled from an internal promoter. However, LTR repair of SIN vectors occurs at an appreciable frequency. Therefore, the extent of this deletion was maximised and the effect on the frequency of the repair examined. The production of lentiviral gene therapy vectors by large-scale transient transfection is both time consuming and technically difficult. Therefore, methods to increase the scale of production without compromising virus titre were developed. This resulted in fewer transfections and less handling of the cells when making virus on a large scale (3-4 L). In order to process the virus on this scale in a single day (i.e. 8 hours), new concentration and purification methods were established. The protocol consisted of low speed centrifugation, 0.45 μm filtration, 750 kDa ultrafiltration, 0.8 μm filtration and ultracentrifugation. However, the use of ultracentrifugation means that this protocol is not amenable to further scale up. Therefore, the replacement of the ultracentrifugation step with anion exchange was investigated. A number of different resins and anion exchange devices were investigated, two of which show promise for large scale purification of HIV-1 derived gene transfer vectors. In an ideal world, HIV-1 derived gene transfer vectors would be produced using stable packaging cell lines engineered to produce the desired virus. However, previous attempts to produce such a cell line with the desired properties have had limited success and have generally used outdated helper systems. Therefore, in an attempt to combine the efficiency advantages of having a single helper plasmid with the safety advantages of expressing each protein separately, a single packaging construct that contained separate transcription units for each of the required proteins was produced. Transcription of cyotoxic proteins was controlled by inducible promoters. Initial results suggest that such a system is technically feasible but that further work is required to optimise the expression of helper functions.
http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1309550
Thesis (Ph.D.) -- School of Paediatrics and Reproductive Health, 2008

Linear covalently closed (LCC) DNA minivectors serve to be superior to conventional circular covalently closed (CCC) plasmid DNA (pDNA) vectors due to enhancements to both transfection efficiency and safety. Specifically, LCC DNA minivectors have a heightened safety profile as insertional mutagenesis is inhibited by covalently closed terminal ends conferring double-strand breaks that cause chromosomal disruption and cell death in the low frequency event of chromosomal integration. The development of a one-step, E. coli based in vivo LCC DNA minivector production system enables facile and efficient production of LCC DNA minivectors referred to as DNA ministrings. This novel in vivo system demonstrates high versatility, generating DNA ministrings catered to numerous potential applications in gene therapy and vaccine development. In the present study, numerous aspects pertaining to the generation of gene therapeutics with LCC DNA ministrings have been explored with relevance to both industry and clinical settings. Through systematic assessment of induction duration, cultivation strategy, and genetic/chemical modifications, the novel in vivo system was optimized to produce high yields of DNA ministrings at ~90% production efficiency. Purification of LCC DNA ministrings using anion exchange membrane chromatography demonstrated rapid, scalable purification of DNA vectors as well as its potential in the separation of different DNA isoforms. The application of a hydrogel-based strong Q-anion exchange membrane, with manipulations to salt gradient, constituted effective separation of parental supercoiled CCC precursor pDNA and LCC DNA. The resulting DNA ministrings were employed for the generation of 16-3-16 gemini surfactant based synthetic vectors and comparative analysis, through physical characterization and in vitro transfection assays, was conducted between DNA ministring derived and CCC pDNA derived lipoplexes. Differences in DNA topology were observed to induce differences in particle size and DNA protection/encapsulation upon lipoplex formation. Lastly, the in vivo DNA minivector production system successfully generated gagV3(BCE) LCC DNA ministrings for downstream development of a HIV DNA-VLP (Virus-like particle) vaccine, thus highlighting the capacity of such system to produce DNA ministrings with numerous potential applications.

Tese de mestrado, Ciências Biofarmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2016
The knowledge of gene regulation led to the emergence of gene therapy as a versatile tool for the prevention and treatment of a variety of human diseases. New strategies for gene manipulation have been developed to date. Due to their innate ability to cross cell membranes and bind DNA in a specific and efficient manner, zinc-finger proteins possess a great potential for gene targeting and regulation. Although, zinc-fingers present the critical limitation of their low specificity to the target cells. Antibody fragments have demonstrated to have high specificity which make them a powerful tool to overcome this limitation presented by zinc-fingers. Within this context, this thesis aims to study the improvement of a previously studied therapeutic strategy of gene manipulation by antibody delivery of zinc-fingers. To validate our strategy and as a proof-of-concept, we choose HIV-1 as the disease model since Acquired Immune Deficiency Syndrome (AIDS) is a disease which represent a major global public health issue. Therefore, we engineered four alternative bispecific proteins of an artificial zinc-finger (KRAB-HLTR3) designed to repress the transcription from the HIV-1 LTR promoter coupled to a CXCR4-specific nanobody (VHH). These proteins were successfully expressed and purified with high yields of soluble protein. Afterwards, we evaluated the proteins specificity and affinity to their targets and we demonstrated that these alternative bispecific proteins bind specifically to HLTR3 binding site in a concentration-dependent manner, similarly to previously studied proteins. We also demonstrated that these bispecific proteins bind specifically to CXCR4 receptor at the surface, similarly to previously studied proteins. Finally, we evaluated the bispecific proteins ability to repress transcription from the HIV-1 LTR promoter. We assessed repression of transcription of a destabilized green fluorescent protein (GFP) reporter and we demonstrated that these constructions repress transcription of GFP gene driven by the HIV-1 LTR promoter in a concentration-dependent manner, like to previously studied proteins. Although results suggest that zinc-finger on the C-terminal promote the binding to the HIV-1 LTR promoter and consequently the protein ability to repress viral transcription. With this in mind, more in vitro studies have to be performed to evaluate the bispecific proteins ability to repress transcription of the HIV-1 genome. In fact, since activation of the HIV-1 LTR promoter lead to expression of the HIV-1 genome, these results suggest that in the presence of the HIV-1 genome, these proteins inhibit the HIV-1 LTR promoter and consequently repress transcription of the HIV-1 genome. Additionally, to evaluate the repression of viral replication by these bispecific proteins, infection assays must be performed in Jurkat cell line or primary CD4+ T-lymphocytes. The infection can be performed with HIV-1 laboratory-adapted strains or HIV-1 primary isolates. In conclusion, results presented in this dissertation demonstrated that these therapeutic proteins improve the antibody delivery strategy to gene manipulation previously developed in our laboratory. In fact, these proteins are a promising tool to be applied in the clinical and might complement other gene-based strategies. Furthermore, these recombinant proteins can be designed and engineered to use in other therapeutic applications.
Nas últimas duas décadas, a terapia génica tem emergido como uma alternativa promissora para o tratamento de uma grande variedade de doenças. Esta terapia consiste na transferência de transgenes para células alvo, promovendo a correção de anormalidades no fenótipo ou genótipo dos pacientes. O crescente conhecimento da regulação génica, assim como da estrutura e função do genoma humano levaram ao aparecimento de novas estratégias para manipulação da expressão génica. O sucesso destas novas estratégias terapêuticas está intimamente relacionado com a escolha de sistemas de entrega de genes eficientes, específicos e não tóxicos. Até ao momento várias estratégias para manipulação génica foram desenvolvidas e testadas. As mais comuns incluem vetores virais e algumas formas de ADN não viral. O uso de vetores virais como veículos de transferência e expressão génica representa um poderoso instrumento, dada a capacidade que os vírus apresentam de penetrar dentro do núcleo das células hospedeiras e explorar a sua maquinaria celular. Entre os mais usados estão os vetores retrovirais, adenovirais e adeno-associados que apesentam alta eficiência de transfecção in vivo. No entanto, a elevada imunogenicidade, o limitante tamanho do transgene e ainda a produção de toxinas são alguns problemas destas estratégias. Relativamente às estratégias não virais, elas incluem inoculação de ADN puro ou encapsulado através de técnicas como microinjeção e electroporação. Os sistemas não virais representam uma importante alternativa aos virais, dada a sua menor imunogenicidade e não apresentarem limitações no tamanho do transgene. Apesar das suas vantagens, estes vetores apresentam uma transfecção menos eficiente comparativamente aos virais. Com o intuito de ultrapassar algumas limitações, quer dos vetores virais como dos não virais, as proteínas dedos de zinco (do inglês, zinc-finger proteins (ZFs)) surgiram como ferramentas versáteis para a terapia génica. Os ZFs apresentam uma capacidade inata para atravessar as membranas celulares e de se ligarem eficientemente e especificamente ao ADN. Desta forma, os ZFs podem ser desenhados para reconhecer uma vasta gama de sequência de ADN de modo a ativar, reprimir, XIV cortar ou colar genes. No entanto, apresentam ainda algumas limitações que devem ser ultrapassadas no futuro, em particular a sua baixa especificidade para as células alvo. Novas estratégias terapêuticas têm sido desenvolvidas de modo a ultrapassar esta limitação dos ZFs. De facto, dada a sua alta especificidade, os anticorpos monoclonais bem como pequenos derivados de anticorpos recombinantes têm demonstrado um enorme potencial para combater este problema dos ZFs. Neste contexto, este projeto científico tem como objetivo melhorar uma estratégia terapêutica de manipulação génica através da entrega de ZFs por anticorpos anteriormente desenvolvida no nosso laboratório. Como prova do conceito, escolhemos como modelo de doença o síndroma de imunodeficiência adquirida (SIDA), uma doença infeciosa de incidência mundial causada pelo vírus da imunodeficiência humana (VIH-1). A infeção pelo VIH-1 é caracterizada por uma supressão do sistema imunitário, levando ao aparecimento de doenças oportunistas. Este vírus tem a capacidade de infetar células CD4+ como é o caso dos linfócitos T e dos macrófagos. No entanto, não só o receptor celular CD4 permite a ligação e consequente entrada do vírus nas células alvo, o receptor de quimiocinas CXCR4 como também o CCR5 determinam também o tropismo celular do vírus. Apesar dos progressos realizados no tratamento da SIDA, especialmente através do uso de fármacos antirretrovirais (HAART), estes não são capazes de erradicar por completo o vírus do organismo. Deste modo, o desenvolvimento de novas estratégias contra o VIH-1, tal como a terapia génica, mostra especial interesse. Com este propósito, foram inicialmente construídas quatro proteínas biespecíficas variantes das já desenvolvidas no nosso laboratório, compostas por um anticorpo recombinante (VHH, também designado por nanobody) desenhado contra o receptor CXCR4 e um ZF com o domínio repressor KRAB desenhado para reprimir a transcrição do genoma do VIH-1 através da sua ligação ao promotor LTR do VIH-1 (KRAB-HLTR3). De forma a avaliar qual a conformação que proporciona uma maior estabilidade e solubilidade à proteína, uma das construções foi desenhada com o KRAB-HLTR3 a N-terminal, similarmente a uma das proteínas já desenvolvidas, enquanto nas outras três foi adicionado a C-terminal. Além disso, para facilitar a libertação do ZF nas células alvo, em duas construções foi introduzida a sequência de clivagem da catepsina B, uma cisteína proteinase lisossomal. Com o mesmo propósito, numa das construções foi introduzida a sequência de clivagem da MMP-9. Por outro lado, foi feita a construção de uma proteína que consiste apenas no KRAB-HLTR3 que foi gentilmente cedida pela C. Cunha-Santos (Laboratório João Gonçalves). Após a construção, todas as proteínas recombinantes foram clonadas no mesmo vector de expressão bacteriano, expressas em E. coli e posteriormente purificadas. Com exceção da construção que possui o sítio de clivagem da MMP-9, todas as outras foram purificadas com sucesso, sendo utilizadas nos ensaios seguintes. Seguidamente, para avaliar a capacidade de ligação de cada proteína biespecífica ao promotor LTR do VIH-1, foram realizados ensaios preliminares de ligação por ELISA, usando uma sequência de oligonucleótidos reconhecida pelo KRAB-HLTR3 como antigénio (sitio de ligação do HLTR3, do inglês HLTR3 binding site). Verificámos que estas proteínas recombinantes ligam especificamente ao sítio de ligação do HLTR3 e que esta ligação é dependente da concentração, o que se assemelha com as proteínas anteriormente estudadas. Sendo que as proteínas são biespecíficas foi necessário avaliar a funcionalidade dos dois domínios funcionais. Com este propósito, um ensaio de citometria de fluxo na linha celular Jurkat E6-1 T foi realizado e verificámos que estas proteínas recombinantes ligam especificamente à superfície do receptor CXCR4, similarmente ao observado com as proteínas já estudadas. De forma a validar a capacidade das proteínas biespecíficas de reprimir a transcrição viral através do promotor LTR do VIH-1, foi avaliada a repressão da transcrição de um gene repórter, nomeadamente a GFP (do inglês green fluorescent protein) conduzido pelo promotor LTR do VIH-1. Ensaios de citometria de fluxo foram realizados na linha celular HeLa-Tat-III/LTR/d1EGFP e verificámos que estas proteínas biespecíficas reprimem a transcrição do gene da GFP e que esta repressão é dependente da concentração, o que se assemelha com as proteínas estudadas anteriormente. Verificámos no entanto que as construções que possuem o KRAB-HLTR3 a C-terminal apresentam uma maior capacidade de repressão da transcrição. Uma vez que a ativação do promotor LTR do VIH-1 leva à expressão do genoma do VIH-1, estes resultados sugerem que na presença do genoma do VIH-1 é possível inibir o promotor LTR e consequentemente reprimir a transcrição do genoma do VIH-1. Simultaneamente, e dado ao facto dos fragmentos de anticorpos exibirem algumas limitações farmacocinéticas, outra estratégia terapêutica foi desenvolvida. Desta forma, construímos três proteínas biespecíficas compostas por um anticorpo monoclonal desenhado contra o receptor HER2 (Trastuzumab, Herceptin®) acoplado a um ZF com o domínio repressor KRAB desenhado para se ligar ao promotor do protooncogene erbB-2/HER-2. Para facilitar a libertação do ZF nas células alvo, numa das construções foi introduzida a sequência de clivagem da catepsina B e noutra construção a sequência de clivagem da MMP-9. Os resultados dos ensaios preliminares de transfecção mostraram que à exceção da construção que possui a sequência da MMP-9, as restantes duas foram construídas e expressas na linha celular HEK293T com sucesso. Em conclusão, os resultados apresentados neste projeto científico demonstraram que estas proteínas biespecíficas melhoram a estratégia terapêutica de manipulação génica anteriormente desenvolvida no nosso laboratório. De facto, estas proteínas biespecíficas apresentam um enorme potencial para serem aplicadas na clinica. Como perspetivas futuras, mais ensaios de citometria de fluxo na linha celular Jurkat E6-1 T são necessários de forma a avaliar a internalização das proteínas biespecíficas via CXCR4. Além disso, para validar que a ligação e a internalização das proteínas ocorre efetivamente via CXCR4, os mesmos ensaios terão de ser realizados na linha celular Jurkat CXCR4 negativas. Por outro lado, para avaliar a influência da catepsina B na libertação do ZF, ensaios funcionais deverão ser realizados. Relativamente à capacidade das proteínas biespecíficas de reprimirem a transcrição do genoma do VIH-1 através da inibição do promotor LTR, terão de ser realizados ensaios in vitro numa linha celular que integre o genoma do VIH-1. Adicionalmente para avaliar a repressão da replicação viral por estas proteínas biespecíficas, ensaios de infeção deverão ser feitos na linha celular Jurkat ou em linfócitos primários.