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CHAPTER 5: DISCUSSION
Cancer is a disorder caused by alterations of the genetic material in normal cells that unbalance the physiological ratio between cell proliferation and programmed cell death (apoptosis) thus leading to uncontrolled cell division. These mutations can be caused by several factors ranging from environment, chemicals exposure and genetic predisposition (Anand et al., 2008). These factors intervene in carcinogenesis, a multistep process caused by a sequential derangement of certain classes of genes involved in cell division, apoptosis and DNA repair. BRCA1 is a tumor suppressor gene involved in cell cycle checkpoints, transcription, protein ubiquitination, apoptosis and DNA repair. Mutations of BRCA1 inactivating the gene have been positively correlated to breast cancer onset. In particular, 80% hereditary breast cancers are associated to BRCA1 or BRCA2 alteration(s). Moreover, women carrying BRCA1 mutations have an 60-80% estimated risk of developing neoplasia (Teng et al., 2008). Development of preventative strategies to counteract genetic predisposition to BRCA1 alteration associated cancer may become a suitable alternative to current intervention methods. Despite many attempts, the most effective remain prophylactic mastectomy and chemoprevention (Rebbeck et al., 2004). Unfortunately, the side effects associated with these approaches, ranging from menopausal symptoms to an increased risk of developing endometrial cancer (Narod et al., 2000) and the serious psychological and social consequences of prophylactic mastectomy leave room to devise and investigate alternative strategies. Among these, substitution or insertion of wild type BRCA genes into mutated cells could stop or impede the initiation of carcinogenesis or block the disorder at its initial stage.
Virus derived vectors are one of the most efficient tools to perform gene delivery into specific target cells. Along evolution viruses have evolved an efficient mechanism to bind a specific cell type (targeting) and release their genetic material inside the cell.
63 Therefore, if appropriately engineered, they can be exploited to deliver the therapeutic gene(s) into appropriated target cells. Among viruses apt to produce viral vectors, lentiviruses are particularly apt for gene therapy, since they can integrate the therapeutic gene into host cell genome and ensure its long-term expression.
From this ground, we thought that vector-mediated delivery of a fully functional BRCA1 gene into cells bearing a defective gene might become a suitable preventative strategy against breast cancer. Delivery will be carried out with a lentiviral vector derived from FIV, chosen because of its close similarity to HIV, one of most effective vectors, but that cannot infect humans thus increasing safeness and potential employment in clinical trials. However, because of natural resistance of human cells to FIV infection, it has been necessary to remodel extensively viral genome and architecture. Since restriction is basically due to low FIV LTRs activity and envelope tropism in human cells, the first step was to replace these two components with the stronger CMVp and with heterologous GPs VSV-G or RD114,. These changes allowed transducing non-feline cells such as human 293T and murine NIH3T3. Even after these changes, however, LA34 maintained a preferential tropism for feline cells and the transduction efficiency of other histotypes remained quite low (Pistello et al., 2007). To overcome this limitation, and based on what already done for similar vectors (Loewen et al., 2003), (Saenz & Poeschla, 2004), we tried to increase transduction of non-feline cells by elongating the gag portion of our vector prototypes (LA34L and LAW34L) from 120 to 311 bp, since previous reports suggested that ψ signal, necessary for incorporating the viral genome into assembling virions, could spread through the first 300 nt of gag. Moreover the WPRE element, important to stabilize the viral RNA genome and transcripts, was inserted downstream the expression cassette as found useful for HIV vectors (Zufferey et al., 1999)(Higashimoto et al., 2007).
With these modifications, the new vector, LAW34, transduced with enhanced efficiency all cell lines tested in this thesis. Importantly, the beneficial effect of WPRE was most evident in NIH-3T3 cells, whose transduction efficiency almost doubled. In contrast, elongation of gag region did not bring any advantage and, in non-feline cells, even led to reduced transduction efficiency. For these reasons LAW34 (having the WPRE element and the shorter gag region) was used for the whole work.
64 One of the main characteristics that make a lentiviral vector safe is that the proteins necessary for viral replication and assembling are provided in more plasmids (thus reducing the risk of recombination) and only the construct expressing the transgene contains the encapsidation signal to permit its packaging into vector particles. To verify that p∆env1, the packaging construct that provides the structural proteins and enzymes for particle formation, was not transferred from packaging cells to transduced cells, we performed a PCR on DNA and RNA extracted from transduced cells to amplify a gag region present only in the packaging construct. Neither gag DNA nor RNA was amplified demonstrating that this packaging construct was not incorporated into assembling virions.
To further increase safety, LAW34 was of SIN type. This was achieved through the complete removal of U3 in 5’ LTR and a deletion of 130 nt in the U3 region at 3’ LTRs. To verify whether LAW34 truly has both LTR inactivated, we tested if the U3 deletion was effectively transferred to the 5’LTR in proviral genome integrated into target cell genome and that this process led to transcription block from both viral promoters. This was done by performing two different PCR on DNA and/or RNA extracted from transduced cells. The first amplification was carried out by extending a portion including the deletion and, as expected, the region amplified was 130 nt shorter than the wild type LTR thus demonstrating translocation of deletion to 5’ LTR. With second PCR we verified the inactivation of viral promoter by amplifying a portion of vector construct immediately downstream the 5’LTR. The negative result confirmed that the translocation led to inactivation of viral promoter in transduced cells. Consequently, once LAW34 integrated into target cells, the only transcribeable region is the expression cassette in which the CMVp drives the expression of the transgene. The presence of an inactive viral promoter in target cells also reduces the risk of insertional mutagenesis also caused by enhanced expression of host cell genome flanking sequences driven by LTRs. Using a SIN vector is therefore important to ensure higher safety levels for an in vivo approach.
Our working strategy, i.e. to delivery wild type BRCA1 with LAW34, was tested on the HCC1937 cell line, chosen as it contains an inactivated BRCA1. These cells, indeed, were isolated and established from a tumor patient with a BRCA1 truncated, non-functional protein (Tomlinson et al., 1998). In a preliminary set of experiments, we assessed the ability of LAW34 to transduce this cell line. For this purpose we used the GFP as gene reporter to analyse the efficiency by FACS analysis and tested two
65 different GPs: the VSV-G, that by using an ubiquitary membrane phospholipid as receptor, confers a wide cell tropism, or RD114/TR, a recombinant GP derived from RD114 feline endogenous virus and optimized for efficient vector entry in target cells (Di Nunzio et al., 2007). RD114/TR, which uses a sodium-dependent neutral amino acid transporter as receptor, was more efficient than VSV-G to transduce murine cells (Pistello et al., 2007), but was ineffective when tested on HCC1937. These findings might be due to the lack of this particular receptor in this cell type rather than to a downstream inhibition. Indeed, when the same vector was coated with VSV-G we achieved 20% transduction of HCC1937, thus demonstrating that once entered into the cytoplasm the RNA vector was free to express the reporter gene. Moreover, the percentage of GFP positive cells did not change throughout over four weeks of observation and upon several freezing and thawing processes indicating that the vector integrates in transduced cells.
Since the percentage of transduced cells was not so high, we tested different transduction protocols to increase the efficiency, i.e. addition of PB, DT and combination of both. DT protocol consists of two sequential transductions performed four hours apart. PB is a cationic polymer that neutralizes charge repulsion between vector particles and sialic acid on the cell surface (Davis et al., 2004). Optimal PB amount was determined by transducing 293T cells with doses ranging from 2 to 10 µg/ml. Best results were obtained with 8 µg/ml that were used in all experiments. The same vector preparation was used to transduce HCC1937, NIH3T3, and 293T with all protocols and the results demonstrated that all procedures increased transduction efficiency to almost forty percent of HCC1937 cells when treated with DT and PB. From these results, DT and PB were used in almost all the following experiments. Once verified that LAW34 can transduce tumor cells and, therefore, that it can be used to deliver transgenes into HCC1937 cells, we started to clone the wild type BRCA1 into LAW34. BRCA1 gene is made of 24 exons extending on 100 kb of human genome. For this reason, we use the entire cDNA of 5700 bp cloned into a commercial plasmid. The unsuccessful effort to insert the entire gene with a singlecloning are probably due to the large size of the gene and, for this reason, the insertion was carried out by performing four subsequent cloning. For this we used selected enzymes with no restriction activity in LAW34 and cutting only once in BRCA1 gene. Through this system, we inserted the whole gene sequence into our vector construct. Each single cloned fragment was verified by screening,
66 sequencing and the complete BRCA1 expression by WB on transfected cells. The higher amount of protein in transfected cells, compared to endogenous BRCA1 protein expressed in mock and naïve cells, demonstrated the functionality of LAW34. Thanks to extensive deletions in LAW34 as compared to wild type FIV, the vector construct is only 2848 bp long, thus permitting to incorporate the entire BRCA1 cDNA and maintain a size length fully compatible with packaging into progeny virions. This was demonstrated by reverse transcriptase real time PCR on RNA extracted from virions released into supernatant of transfected cells. Further, to avoid amplification of residual DNA into supernatant, the extracted nucleic acid was previously treated with DNAse and primers and probe were designed on the heterologous element WPRE contained only into the vector. For comparison, real time PCR was also performed on LAW34 and LAW34-GFP sizing 2848 bp and 3567 bp, respectively. The results showed that RNA copies are similar for all vector tested indicating similar efficiency of packaging for all constructs and demonstrating that LAW34 can carry large transgenes. Further proof of efficient vehiculation of BRCA was obtained by demonstrating BRCA1 expression in transduced HCC1937 by FACS analysis.
An important domain of BRCA1 is localized at the C-terminus, therefore even if the mutated BRCA1 expressed by HCC1937 only lacks a stretch of 34 aa within this domain, the protein is non-functional. Since the difference between wild type and this as well as other mutated forms is small, there are no commercial Abs able to distinguish between wild type and mutated proteins. For this reason, both mock and transduced cells were nearly 100% positive when tested with a fluorescent anti-BRCA1 mAb.
Although percent fluorescent cells was similar, when mock and transduced HCC1937 were analyzed for intensity of fluorescence the latter showed a much higher intensity compared to the mock cells.
This result demonstrates that wild type BRCA1 is expressed in transduced cells and, therefore, that the vector construct prepared is able to deliver and correctly express BRCA1 in transduced cells.
Delivery and expression aside, a prerequisite for the development of an effective protocol is that the transgene preserves the functional activity and is able to correct the genetic defect. This was tested with a RRR test. Since BRCA1 also repairs DNA damages, we exploited this feature to assess its activity by artificially damaging cellular DNA of BRCA1 transduced cells by γ-irradiation. To facilitate monitoring of
67 cell recovery and growth upon exposition to γ-rays, we purposely developed a bicistronic vector co-expressing BRCA1 and GFP protein. Co-delivery and expression of both transgenes into a unique vector can be achieved with two strategies: cloning two independent promoters for each transgenes, or inserting an IRES between the two transgenes. IRES is a particular region with highly structured RNA which mediates cap-independent translation initiation. This function is encoded in conserved structural motifs whose secondary structure is recognized and bound by ribosomes, which then initiate translation of downstream gene (Ngoi et al., 2004). Since two promoters in same vector could interfere each other, to obtain a bicistronic vector we used the IRES from encephalomyocarditis virus, inserted downstream BRCA1 and upstream GFP.
Once the vector BRCA1-IRES-GFP was produced, we verified its functionality by monitoring GFP with fluorescent microscopy and FACS analysis on 293T cells. Transfected cells successfully expressed GFP but percent of fluorescent cells and intensity of fluorescence were lower than those obtained with LAW34-GFP. This was expected since expression of the downstream gene occurs at lower efficiency compared to the upstream gene (Ngoi et al., 2004) , especially if the upstream gene is of large size as for BRCA1. BRCA1-IRES-GFP encapsidation was evaluated as we did for LAW34-BRCA1. BRCA1-IRES-GFP RNA copies resulted similar to those obtained with LAW34-BRCA1, LAW34 and LAW34-GFP demonstrating that even if vector construct is about 1 kb longer than the FIV genome nevertheless it could be packaged into assembling particles as for shorter vectors. We also verified if BRCA1-IRES-GFP could express both transgenes in transduced HCC1937 by FACS analysis. As for transfected cells, percent of GFP positive HCC1937 cells and intensity of fluorescence were lower compared to that obtained with LAW34-GFP but the presence of fluorescent cells confirmed effective encapsidation and ability of BRCA1-IRES-GFP to transduce HCC1937. To monitor BRCA1 expression, we performed the same FACS analysis carried out for LAW34-BRCA1. Also in these cells the intensity of fluorescence of labeled transduced HCC1937 was higher than labeled mock cells, thus demonstrating wild type BRCA1 expression in cells transduced with bicistronic vector. In all these results indicate that our vector can deliver up to 7 kb of heterologous material and express more than one gene in transduced cells.
Finally we used BRCA1-IRES-GFP in parallel with LAW34-GFP to perform the RRR test. Transduction of HCC1937 with BRCA1-IRES-GFP and LAW34-GFP allows to monitor cell
68 growth after irradiation and to assess the ability to repair DNA damages by monitoring cell growth of GFP-positive cells.
A crucial parameter for this test is the irradiation dose. To assess BRCA1 activity it is essential to use a sub-lethal dose sufficient to induce heavy damages to cellular genome but not so to start apoptosis. To choose the most suitable irradiation dose we set up preliminary experiments by damagingHCC1937 cells with increasing irradiation doses and monitoring cell viability and growth. The results showed that the doses ranging from 1 to 2.3 Gy of irradiation caused a transient damage that reduced cell viability and growth for 2-3 days after which they quickly recovered and from day 5 onward showed parameters similar to naïve cells. In contrast, doses over 4 Gy progressively caused heavy damages eventually leading to cell death. Therefore, we chose 2.3 Gy, dose at which cells suffered a sub-lethal, damage that lasted for several days.
LAW34-GFP and BRCA1-IRES-GFP transduced HCC1937 cells were exposed to 2.3 Gy of γ-rays and monitored for GFP positive cells. Since mock cells irradiated with same dose fully recovered within 5 days, transduced and irradiated cells were checked every day for one week. Upon irradiation, BRCA1-IRES-GFP transduced cells grew better and five times faster than LAW34-GFP cells. This finding indicates that wild type BRCA1 helps cells to recover faster probably as a result of a more efficient DNA repair mechanisms compared to HCC1937 cells expressing the defective gene. To further validate the strategy we evaluate whether LAW34-BRCA1 can transduce primary cells, i.e. the natural target for in vivo application. Although HCC1937 cells derive from a patient with breast cancer, it is very likely that following prolonged propagation in culture, the cells have lost most properties and features typically observed in ex vivo cells, i.e. cells directly derived from a tissue. Thus not wistanding the encouraging results obtained with the cell line, it was important to test whether LAW34 can also transduce primary epithelial cells derived from a breast cancer patient. To this aim, primary epithelial cells with a mutated BRCA1 were obtained from a patient subjected to mastectomy at Dr. Caligo’s laboratory (Department of Oncology, Division of Surgical, Molecular and Ultrastructural Pathology, University of Pisa). Cells were transduced with the bicistronic vector and LAW34-GFP as a control, and monitored for GFP expression by FACS analysis. Despite the primary colture was heavily contaminated by fibroblasts, epithelial and fibroblast cells can be easily distinguished by visual inspection and FASC analysis thus allowing to monitor the
69 ability of our vectors to transduce one or both populations. Flow cytometry analysis demonstrated that both vectors preferentially transduced the epithelial cells and with efficiency comparable to the HCC1937 cell line. In contrast, percent of transduced fibroblasts was extremely low indicating that these cells were somehow refractory. However, since breast cancer originates from the epithelial cells the result further emphasize the suitability of this vector for this strategy.
In conclusion, this work demonstrates that our vector is able to deliver and express a functional BRCA1 that rescues lost gene activity in tumor cells. In addition, LAW34 demonstrated able to vehiculate up to 7,000 bp of heterologous material, and is therefore applicable to deliver very long genes in specific cells. Finally, LAW34 is also able to deliver and express more transgenes and, as recently published (Pistello et al., 2007), have a different spectrum of targetable cells depending on the coat proteins used for pseudotyping. Because of their hitherto unmatched performances, most gene delivery approaches have been devised with HIV derived vectors. The results showed here demonstrate that our FIV vector is valid alternative to HIV as regards delivering properties and, due to higher safety standards, has fewer impediments for in vivo application. Finally, the effective replacement of BRCA1 shows that this strategy is potentially exploitable to set up novel preventative approaches against human breast cancer.
