Development of a Palliative Care Approach for Primary Progressive Aphasia: My Experience as a Person Living With This Rare Disorder.

Frontotemporal disorders are a group of rare young-onset dementias for which there is no cure, nor is there any way to slow the underlying progressive brain degeneration. To date those affected have typically received very little, if any, follow-up care after diagnosis, particularly in the early stages of their disease. I have received a clinical diagnosis, supported by imaging, of primary progressive aphasia, a form of frontotemporal degeneration characterized in the initial phase by progressive impairment of language ability. From the onset, I have been fortunate to receive excellent ongoing palliative care from a multidisciplinary team, some of whom had never previously seen anyone with this disorder. My quality of life has been enhanced by an evolving range of creative strategies and adaptations targeted to my deficits as they have arisen. In this paper, I discuss my experience of the process underlying this personalized plan, which serves as a paradigm for the development of novel palliative care approaches for people living with rare disorders, both neurodegenerative diseases and other conditions.

My experience of living with nonfluent/agrammatic variant primary progressive aphasia: Challenges, compensatory strategies and adaptations.

BACKGROUND: Primary progressive aphasia (PPA) is a rare neurodegenerative brain disorder characterized by declining language ability. There is currently no way to reverse or slow the course of the progressive brain degeneration, nor is there a cure for PPA. Throughout the course of the disease, any treatment must therefore be palliative in nature and should be designed to manage symptoms and improve the quality of life of the affected person. There is little information in the medical literature about strategies to make meaningful improvements to the quality of life of people with PPA written from the perspective of those living with this condition. AIMS: I have a clinical diagnosis of the nonfluent/agrammatic variant of PPA (nfvPPA), supported by imaging. In this report I discuss my experience of the progressive loss of language and communication skills, and detail the challenges I have been facing. I also describe how my quality of life has been enhanced by the early initiation of treatment focusing on communication strategies targeted to my specific impairments and designed to support my individual interests and goals. METHODS & PROCEDURES: I was fortunate to obtain an early diagnosis from a cognitive neurologist experienced with PPA. From the onset of my language difficulties, I have received excellent personalized care from a multidisciplinary medical team including speech-language pathologists, a cognitive neurologist and other doctors. MAIN CONTRIBUTIONS: My life during the early stage of nfvPPA has been enriched by personalized care focused on supporting the particular activities, interests and goals that are most important and meaningful to me. As my disease has progressed, I have benefited from an evolving range of strategies and adaptations targeted to the specific deficits in the areas of speaking, writing and reading that I have been facing at any given time. In addition, I have adopted methods to enhance the benefit of these language-directed strategies. And I have been employing evidence-based approaches that improve general brain health and thereby indirectly support my language. CONCLUSIONS & IMPLICATIONS: My experience represents a model for the personalized care of people in the early stage of nfvPPA. WHAT THIS PAPER ADDS: What is already known on the subject There is minimal information in the medical literature describing the subjective experience of a person with PPA. There is little information in the medical literature about strategies to make meaningful improvements to the quality of life of people in the early stage of PPA. What this paper adds to existing knowledge I have a clinical diagnosis of nfvPPA, supported by imaging. In this paper I give a first-person account of my experience of the progressive loss of language and communication skills, and I detail the challenges I have been facing. I describe how my quality of life during the early stage of nfvPPA has been enhanced by an evolving range of strategies and adaptations tailored to my speech and language deficits as they have arisen. These compensatory strategies have focused on supporting the particular activities, interests and goals that are most important and meaningful to me. What are the potential or actual clinical implications of this work? The description of my subjective experience of the progressive loss of language and communication skills offers insight for speech-language pathologists, neurologists and other professionals involved in the clinical care of people in the early stage of nfvPPA. My experience represents a model for the personalized clinical care of people in the early stage of this disorder.

My experience of living with nonfluent/agrammatic variant primary progressive aphasia: Adaptations and strategies to improve quality of life.

Primary progressive aphasia is a major clinical presentation of frontotemporal lobar degeneration and is a young-onset disorder characterized by deteriorating language skills. There is currently no cure for primary progressive aphasia, nor is it possible to slow the course of the underlying progressive brain degeneration. Hence the chief goal of treatment is palliative. Although the inability to employ language at one's previous level represents a significant functional impairment for those affected, there is a dearth of information about how to make meaningful improvements to the quality of life of people in the early stages of primary progressive aphasia. I have a clinical diagnosis, supported by imaging, of the nonfluent/agrammatic variant of primary progressive aphasia and am under the care of a multidisciplinary medical team. This report is based on my ongoing experience and describes the development and implementation of an evolving set of targeted strategies and adaptations designed to enhance the quality of life of a person in the early stages of this disorder.

Adaptation to Early-Stage Nonfluent/Agrammatic Variant Primary Progressive Aphasia: A First-Person Account.

Primary progressive aphasia (PPA) is a young-onset neurodegenerative disorder characterized by declining language ability. The nonfluent/agrammatic variant of PPA (PPA-G) has the core features of agrammatism in language production and effortful, halting speech. As with other frontotemporal spectrum disorders, there is currently no cure for PPA, nor is it possible to slow the course of progression. The primary goal of treatment is therefore palliative in nature. However, there is a paucity of published information about strategies to make meaningful improvements to the quality of life of people with PPA, particularly in the early stages of the disease where any benefit could most be appreciated by the affected person. This report describes a range of strategies and adaptations designed to improve the quality of life of a person with early-stage PPA-G, based on my experience under the care of a multidisciplinary medical team.

Expression of osteoprotegerin from a replicating adenovirus inhibits the progression of prostate cancer bone metastases in a murine model.

Metastatic involvement of the skeleton is a frequent consequence of advanced prostate cancer. These skeletal metastases cause a number of debilitating complications and are refractory to current treatments. New therapeutic options are being explored, including conditionally replicating adenoviruses (CRAds). CRAds are engineered to selectively replicate in and destroy tumor cells and can be 'armed' with exogenous transgenes for enhanced potency. We hypothesized that a CRAd armed with osteoprotegerin (OPG), an inhibitor of osteoclastogenesis, would inhibit the progression of prostate cancer bone metastases by directly lysing tumor cells and by reducing osteoclast activity. Although prostate cancer bone metastases are predominantly osteoblastic in nature, increased osteoclast activity is critical for the growth of these lesions. Ad5-Δ24-sOPG-Fc-RGD is a CRAd that carries a fusion of the ligand-binding domains of OPG and the Fc region of human IgG1 in place of the viral E3B genes. To circumvent low tumor cell expression of the native adenoviral receptor, an arginine-glycine-aspartic acid (RGD) peptide insertion within the viral fiber knob allows infection of cells expressing α(v) integrins. A 24-base pair deletion (Δ24) within viral E1A limits replication to cells with aberrant retinoblastoma cell cycle regulator/tumor suppressor expression. We have confirmed that Ad5-Δ24-sOPG-Fc-RGD replicates within and destroys prostate cancer cells and, in both murine and human coculture models, that infection of prostate cancer cells inhibits osteoclastogenesis in vitro. In a murine model, progression of advanced prostate cancer bone metastases was inhibited by treatment with Ad5-Δ24-sOPG-Fc-RGD but not by an unarmed control CRAd.

A tumor-stroma targeted oncolytic adenovirus replicated in human ovary cancer samples and inhibited growth of disseminated solid tumors in mice.

Targeting the tumor stroma in addition to the malignant cell compartment is of paramount importance to achieve complete tumor regression. In this work, we modified a previously designed tumor stroma-targeted conditionally replicative adenovirus (CRAd) based on the SPARC promoter by introducing a mutated E1A unable to bind pRB and pseudotyped with a chimeric Ad5/3 fiber (Ad F512v1), and assessed its replication/lytic capacity in ovary cancer in vitro and in vivo. AdF512v1 was able to replicate in fresh samples obtained from patients: (i) with primary human ovary cancer; (ii) that underwent neoadjuvant treatment; (iii) with metastatic disease. In addition, we show that four intraperitoneal (i.p.) injections of 5 × 10(10) v.p. eliminated 50% of xenografted human ovary tumors disseminated in nude mice. Moreover, AdF512v1 replication in tumor models was enhanced 15-40-fold when the tumor contained a mix of malignant and SPARC-expressing stromal cells (fibroblasts and endothelial cells). Contrary to the wild-type virus, AdF512v1 was unable to replicate in normal human ovary samples while the wild-type virus can replicate. This study provides evidence on the lytic capacity of this CRAd and highlights the importance of targeting the stromal tissue in addition to the malignant cell compartment to achieve tumor regression.

A simplified method for the generation of human osteoclasts in vitro.

Osteoclasts are large, multinucleated cells responsible for the resorption of mineralized bone matrix. These cells are critical players in the bone turnover involved in bone homeostasis. Osteoclast activity is connected to the establishment and expansion of skeletal metastases from a number of primary neoplasms. Thus, the formation and activation of osteoclasts is an area of research with many potential avenues for clinical translation. Past studies of osteoclast biology have utilized primary murine cells cultured in vitro. Recently, techniques have been described that involve the generation of osteoclasts from human precursor cells. However, these protocols are often time-consuming and insufficient for generating large numbers of osteoclasts. We therefore developed a simplified protocol by which human osteoclasts may be easily and reliably generated in large numbers in vitro. In this study, osteoclasts were differentiated from bone marrow cells that had been aliquotted and frozen. Cells were generated by culture with recombinant macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). Both human and murine RANKL were shown to efficiently generate osteoclasts, although higher concentrations of murine RANKL were required. Formation of osteoclasts was demonstrated qualitatively by tartrate-resistant acid phosphatase (TRAP) staining. These cells were fully functional, as confirmed by their ability to form resorption pits on cortical bone slices. Functional human osteoclasts can be difficult to generate in vitro by current protocols. We have demonstrated a simplified system for the generation of human osteoclasts in vitro that allows for large numbers of osteoclasts to be obtained from a single donor.

Conditionally replicating adenovirus expressing TIMP2 increases survival in a mouse model of disseminated ovarian cancer.

Ovarian cancer remains difficult to treat mainly due to presentation of the disease at an advanced stage. Conditionally-replicating adenoviruses (CRAds) are promising anti-cancer agents that selectively kill the tumor cells. The present study evaluated the efficacy of a novel CRAd (Ad5/3-CXCR4-TIMP2) containing the CXCR4 promoter for selective viral replication in cancer cells together with TIMP2 as a therapeutic transgene, targeting the matrix metalloproteases (MMPs) in a murine orthotopic model of disseminated ovarian cancer. An orthotopic model of ovarian cancer was established in athymic nude mice by intraperitonal injection of the human ovarian cancer cell line, SKOV3-Luc, expressing luciferase. Upon confirmation of peritoneal dissemination of the cells by non-invasive imaging, mice were randomly divided into four treatment groups: PBS, Ad-ΔE1-TIMP2, Ad5/3-CXCR4, and Ad5/3-CXCR4-TIMP2. All mice were imaged weekly to monitor tumor growth and were sacrificed upon reaching any of the predefined endpoints, including high tumor burden and significant weight loss along with clinical evidence of pain and distress. Survival analysis was performed using the Log-rank test. The median survival for the PBS cohort was 33 days; for Ad-ΔE1-TIMP2, 39 days; for Ad5/3-CXCR4, 52.5 days; and for Ad5/3-CXCR4-TIMP2, 63 days. The TIMP2-armed CRAd delayed tumor growth and significantly increased survival when compared to the unarmed CRAd. This therapeutic effect was confirmed to be mediated through inhibition of MMP9. Results of the in vivo study support the translational potential of Ad5/3-CXCR4-TIMP2 for treatment of human patients with advanced ovarian cancer.

Conditionally replicating adenovirus expressing TIMP2 for ovarian cancer therapy.

PURPOSE: Current treatments for ovarian cancer have limited therapeutic outcomes due to advanced stage of the disease at diagnosis. Among new therapies, conditionally replicating adenoviruses (CRAds), designed to selectively lyse cancer cells, hold promise. In clinical trials, CRAds exhibited limited efficacy thus far. Second-generation CRAds are being developed to express a therapeutic protein to enhance antitumor efficacy. One attractive target in the tumor microenvironment is the matrix metalloproteinases (MMPs) that degrade the extracellular matrix, and are upregulated in ovarian cancer. Tissue inhibitor of metalloproteinase 2 (TIMP2) is an endogenous inhibitor of MMPs. The present study developed and evaluated a novel CRAd (Ad5/3-CXCR4-TIMP2) for ovarian cancer therapy. EXPERIMENTAL DESIGN: A targeted CRAd, Ad5/3-CXCR4-TIMP2 was developed using the CXCR4 promoter for enhanced replication, and expressing the TIMP2 transgene. The efficacy of this armed CRAd was determined in both established human ovarian cancer cell lines and in primary ovarian tumor samples. RESULTS: Ad5/3-CXCR4-TIMP2 mediated expression of functional TIMP2, as demonstrated by the inhibition of MMP activity. In addition, arming with TIMP2 did not inhibit viral replication or oncolytic potency, as the TIMP2-armed viruses showed enhanced killing of cancer cells when compared to the unarmed viruses. We also examined viral replication in primary ovarian cancer tissues obtained from patients with stage III and IV ovarian cancer. In four of the five tumor samples, Ad5/3-CXCR4-TIMP2 revealed a 21- to 89-fold increase in replication when compared to the Ad5/3 virus. CONCLUSION: Results support the translational potential of Ad5/3-CXCR4-TIMP2 for treatment of patients with advanced ovarian cancer.

Ex vivo transfer of the Hoxc-8-interacting domain of Smad1 by a tropism-modified adenoviral vector results in efficient bone formation in a rabbit model of spinal fusion.

STUDY DESIGN: Ex vivo gene transfer for spinal fusion. OBJECTIVE: This study aimed to evaluate ex vivo transfer of the nuclear-localized Hoxc-8-interacting domain of Smad1 (termed Smad1C) to rabbit bone marrow stromal cells (BMSCs) by a tropism-modified human adenovirus serotype 5 (Ad5) vector as a novel therapeutic approach for spinal fusion. SUMMARY OF BACKGROUND DATA: Novel approaches are needed to improve the success of bone union after spinal fusion. One such approach is the ex vivo transfer of a gene encoding an osteoinductive factor to BMSCs which are subsequently reimplanted into the host. We have previously shown that heterologous expression of the Hoxc-8-interacting domain of Smad1 in the nuclei of osteoblast precursor cells is able to stimulate the expression of genes related to osteoblast differentiation and induce osteogenesis in vivo. Gene delivery vehicles based on human Ad5 are well suited for gene transfer for spinal fusion because they can mediate high-level, short-term gene expression. However, Ad5-based vectors with native tropism poorly transduce BMSCs, necessitating the use of vectors with modified tropism to achieve efficient gene transfer. METHODS: The gene encoding Smad1C was transferred to rabbit BMSCs by an Ad5 vector with native tropism or a vector retargeted to alphav integrins, which are abundantly expressed on rabbit BMSCs. Transduced BMSCs were maintained in osteoblastic differentiation medium for 30 days. Alkaline phosphatase activity was determined and cells stained for calcium deposition. As positive controls for osteogenesis, we used Ad5 vectors expressing bone morphogenetic protein 2. As negative controls, BMSCs were mock-transduced or transduced with an Ad5 vector expressing beta-galactosidase. In an immunocompetent rabbit model of spinal fusion, transduced BMSCs were coated onto absorbable gelatin sponge and implanted between decorticated transverse processes L6 and L7 of 8-week-old female New Zealand white rabbits. Animals were killed 4 weeks after implantation of the sponges, the fusion masses harvested and the area of new bone quantified using image analysis software. RESULTS: The Smad1C-expressing tropism-modified Ad5 vector mediated a significantly higher level of alkaline phosphatase activity and calcium deposition in transduced rabbit BMSCs than all other vectors. The rabbit BMSCs transduced ex vivo with the Smad1C-expressing tropism-modified Ad5 vector mediated a greater amount of new bone formation than BMSCs transduced with any other vector. CONCLUSIONS: Delivery of the Smad1C gene construct to BMSCs by an alphav integrin-targeted Ad5 vector shows promise for spinal fusion and other applications requiring the formation of new bone in vivo.

Novel infectivity-enhanced oncolytic adenovirus with a capsid-incorporated dual-imaging moiety for monitoring virotherapy in ovarian cancer.

We sought to develop a cancer-targeted, infectivity-enhanced oncolytic adenovirus that embodies a capsid-labeling fusion for noninvasive dual-modality imaging of ovarian cancer virotherapy. A functional fusion protein composed of fluorescent and nuclear imaging tags was genetically incorporated into the capsid of an infectivity-enhanced conditionally replicative adenovirus. Incorporation of herpes simplex virus thymidine kinase (HSV-tk) and monomeric red fluorescent protein 1 (mRFP1) into the viral capsid and its genomic stability were verified by molecular analyses. Replication and oncolysis were evaluated in ovarian cancer cells. Fusion functionality was confirmed by in vitro gamma camera and fluorescent microscopy imaging. Comparison of tk-mRFP virus to single-modality controls revealed similar replication efficiency and oncolytic potency. Molecular fusion did not abolish enzymatic activity of HSV-tk as the virus effectively phosphorylated thymidine both ex vivo and in vitro. In vitro fluorescence imaging demonstrated a strong correlation between the intensity of fluorescent signal and cytopathic effect in infected ovarian cancer cells, suggesting that fluorescence can be used to monitor viral replication. We have in vitro validated a new infectivity-enhanced oncolytic adenovirus with a dual-imaging modality-labeled capsid, optimized for ovarian cancer virotherapy. The new agent could provide incremental gains toward climbing the barriers for achieving conditionally replicated adenovirus efficacy in human trials.

Targeting of adenovirus serotype 5 pseudotyped with short fiber from serotype 41 to c-erbB2-positive cells using bispecific single-chain diabody.

The purpose of the current study was to alter the broad native tropism of human adenovirus for virus targeting to c-erbB2-positive cancer cells. First, we engineered a single-chain antibody (scFv) against the c-erbB2 oncoprotein into minor capsid protein IX (pIX) of adenovirus serotype 5 (Ad5) in a manner commensurate with virion integrity and binding to the soluble extracellular c-erbB2 domain. To ablate native viral tropism and facilitate binding of the pIX-incorporated scFv to cellular c-erbB2, we replaced the Ad5 fiber with the Ad41 short (41s) fiber devoid of all known cell-binding determinants. The resultant Ad5F41sIX6.5 vector demonstrated increased cell binding and gene transfer as compared to the Ad5F41s control; however, this augmentation of virus infectivity was not c-erbB2 specific. Incorporation of a six-histidine (His(6)) peptide into the C-terminus of the 41s fiber protein resulted in markedly increased Ad5F41s6H infectivity in 293AR cells, which express a membrane-anchored scFv against the C-terminal oligohistidine tag, as compared to the Ad5F41s vector and the parental 293 cells. These data suggested that a 41s-fiber-incorporated His(6) tag could serve for attachment of an adapter protein designed to guide Ad5F41s6H infection in a c-erbB2-specific manner. We therefore engineered a bispecific scFv diabody (scDb) combining affinities for both c-erbB2 and the His(6) tag and showed its ability to provide up to 25-fold increase of Ad5F41s6H infectivity in c-erbB2-positive cells. Thus, Ad5 fiber replacement by a His(6)-tagged 41s fiber coupled with virus targeting mediated by an scDb adapter represents a promising strategy to confer Ad5 vector tropism for c-erbB2-positive cancer cells.

Evaluation of E1A double mutant oncolytic adenovectors in anti-glioma gene therapy.

Malignant glioma, in particular glioblastoma multiforme (GBM), represents one of the most devastating cancers currently known and existing treatment regimens do little to change patient prognosis. Conditionally replicating adenoviral vectors (CRAds) represent attractive experimental anti-cancer agents with potential for clinical application. However, early protein products of the wild type adenovirus backbone--such as E1A--limit CRAds' replicative specificity. In this study, we evaluated the oncolytic potency and specificity of CRAds in which p300/CPB and/or pRb binding capacities of E1A were ablated to reduce non-specific replicative cytolysis. In vitro cytopathic assays, quantitative PCR analysis, Western blot, and flow cytometry studies demonstrate the superior anti-glioma efficacy of a double-mutated CRAd, Ad2/24CMV, which harbors mutations that reduce E1A binding to p300/CPB and pRb. When compared to its single-mutated and wild type counterparts, Ad2/24CMV demonstrated attenuated replication and cytotoxicity in representative normal human brain while displaying enhanced replicative cytotoxicity in malignant glioma. These results have implications for the development of double-mutated CRAd vectors for enhanced GBM therapy.

An oncolytic adenoviral vector carrying the tyrosinase promoter for glioma gene therapy.

Targeting gene expression to cancer cells remains a challenge for the development of gene and viral therapy for gliomas. Recent studies have highlighted transcriptional targeting as one of the possible solutions to overcome this limitation. In this context, melanoma associated antigens (MAAs) are usually over-expressed in brain tumors in comparison to normal brain tissue. For this reason, we investigated the use of the tyrosinase promoter as a transcriptional element to target oncolytic therapy for gliomas. Tyrosinase mRNA expression was evaluated by qRT-PCR in normal human brain tissue as well as in human glioma specimens. We found that this gene was significantly over-expressed in glioma cell lines and in primary glioma samples. Tyrosinase expression correlated with the grade of the tumor (p-value range: 0.05-0.001). Furthermore, transfection of several cell cultures with human and mouse tyrosinase promoters driving a luciferase reporter gene confirmed the activity of this promoter in mouse and human cells. To evaluate whether tyrosinase-activated conditionally replicative adenoviruses (CRAds) could induce toxicity in glioma cells, two vectors (Ad h/m and Ad24TYR) were tested in a mouse glioma model. C57BL/6 mice underwent intracranial injection of tumor cell line GL261. Survival was used to evaluate efficacy of the tested vectors. Mice receiving 1 x 10(9) MOI of Ad h/m and Ad24TYR following intracranial tumor implants had a median survival of 46+/-3 days (p<0.05); in contrast, those treated with medium had a median survival of 31+/-2 days. These results suggest that injection of tyrosinase CRAds leads to prolongation of survival in mice with experimental brain tumors. The tyrosinase promoter stands as a proof of principle of the potential use of MAA over-expression patterns for targeting novel anti-glioma therapies.

Adenoviral vectors for gene therapy.

Vectors based on human adenovirus serotypes 2 (Ad2) and 5 (Ad5) of species C possess a number of features that have favored their widespread employment for gene delivery both in vitro and in vivo. However, the use of recombinant Ad2- and Ad5-based vectors for gene therapy also suffers from a number of disadvantages. These vectors possess the tropism of the parental viruses, which infect all cells that possess the appropriate surface receptors, precluding the targeting of specific cell types. Conversely, some cell types that represent important targets for gene transfer express only low levels of the cellular receptors, which lead to inefficient infection. Another major disadvantage of Ad2- and Ad5-based vectors in vivo is the elicitation of both an innate and an acquired immune response. Considerable attention has therefore been focused on strategies to overcome these limitations, thereby permitting the full potential of adenoviral vectors to be realized.

Mesenchymal stem cells as a vehicle for targeted delivery of CRAds to lung metastases of breast carcinoma.

PURPOSE: Alternative and complementary therapeutic strategies need to be developed for metastatic breast cancer. Virotherapy is a novel therapeutic approach for the treatment of cancer in which the replicating virus itself is the anticancer agent. However, the success of virotherapy has been limited due to inefficient virus delivery to the tumor site. The present study addresses the utility of human mesenchymal stem cells (hMSCs) as intermediate carriers for conditionally replicating adenoviruses (CRAds) to target metastatic breast cancer in vivo. EXPERIMENTAL DESIGN: HMSC were transduced with CRAds. We used a SCID mouse xenograft model to examine the effects of systemically injected CRAd loaded hMSC or CRAd alone on the growth of MDA-MB-231 derived pulmonary metastases (experimental metastases model) in vivo and on overall survival. RESULTS: Intravenous injection of CRAd loaded hMSCs into mice with established MDA-MB-231 pulmonary metastatic disease homed to the tumor site and led to extended mouse survival compared to mice treated with CRAd alone. CONCLUSION: Injected hMSCs transduced with CRAds suppressed the growth of pulmonary metastases, presumably through viral amplification in the hMSCs. Thus, hMSCs may be an effective platform for the targeted delivery of CRAds to distant cancer sites such as metastatic breast cancer.

Combining high selectivity of replication via CXCR4 promoter with fiber chimerism for effective adenoviral oncolysis in breast cancer.

Conditionally replicative adenoviruses (CRAds) represent novel therapeutic agents that have been recently applied in the context of breast cancer therapy. However, deficiencies in the ability of the adenovirus to infect target tumor cells and to specifically replicate within the tumor target represent key deficiencies preventing the realization of the full potential of this therapeutic approach. Minimal expression of the adenovirus serotype 5 (Ad5) receptor CAR (coxsackie and adenovirus receptor) on breast cancer cells represents a major limitation for Ad5-based virotherapy. Genetic fiber chimerism is a method to alter the tropism of Ad5-based CRAds to achieve CAR-independent infectivity of tumor cells. Here, we describe the use of a CRAd with cancer specific transcriptional control of the essential Ad5 E1A gene using the human CXCR4 gene promoter. We further modified the fiber protein of this agent by switching the knob domain with that of the adenovirus serotype 3. The oncolytic activity of this 5/3 fiber-modified CRAd was studied in breast cancer cell lines, primary breast cancer and human liver tissue slices from patients, and in a xenograft breast cancer mouse model. This infectivity enhanced CRAd agent showed improved replication and killing in breast cancer cells in vitro and in vivo with a remarkable specificity profile that was strongly attenuated in nonbreast cancer cells, as well as in normal human breast and liver tissues. In conclusion, utilization of a CRAd that combined infectivity enhancement strategies and transcriptional targeting improved the CRAd-based antineoplastic effects for breast cancer therapy.

Tissue inhibitor of metalloproteinases-2 improves antitumor efficacy of a replicating adenovirus in vivo.

Clinical studies of replicating adenoviruses for the treatment of cancer have demonstrated their safety but have yielded disappointing results, indicating the need for new strategies to improve their efficacy. We hypothesized that the efficacy of a replicating adenovirus could be improved by expression of tissue inhibitor of metalloproteinases-2 (TIMP-2), a 21-kDa unglycosylated secretory protein. TIMP-2 specifically inhibits the active forms of a number of matrix metalloproteinases (MMPs) that play a role in the degradation of basement membranes and the extracellular matrix and are therefore involved in the control of the growth, invasion and metastasis of tumor cells, as well as angiogenesis. In addition, TIMP-2 can abrogate tumor growth and angiogenesis by a variety of mechanisms independent of MMP inhibition. In this study, we demonstrate that expression of TIMP-2 enhanced the antitumor efficacy of a replicating adenovirus in vivo, by reducing both tumor growth and angiogenesis.

An adenovirus serotype 5 vector with fibers derived from ovine atadenovirus demonstrates CAR-independent tropism and unique biodistribution in mice.

Many clinically important tissues are refractory to adenovirus (Ad) infection due to negligible levels of the primary Ad5 receptor the coxsackie and adenovirus receptor CAR. Thus, development of novel CAR-independent Ad vectors should lead to therapeutic gain. Ovine atadenovirus type 7, the prototype member of genus Atadenovirus, efficiently transduces CAR-deficient human cells in vitro, and systemic administration of OAdV is not associated with liver sequestration in mice. The penton base of OAdV7 does not contain an RGD motif, implicating the long-shafted fiber molecule as a major structural dictate of OAdV tropism. We hypothesized that replacement of the Ad5 fiber with the OAdV7 fiber would result in an Ad5 vector with CAR-independent tropism in vitro and liver "detargeting" in vivo. An Ad5 vector displaying the OAdV7 fiber was constructed (Ad5Luc1-OvF) and displayed CAR-independent, enhanced transduction of CAR-deficient human cells. When administered systemically to C57BL/6 mice, Ad5Luc1-OvF reporter gene expression was reduced by 80% in the liver compared to Ad5 and exhibited 50-fold higher gene expression in the kidney than the control vector. To our knowledge, this is the first report of a fiber-pseudotyped Ad vector that simultaneously displays decreased liver uptake and a distinct organ tropism in vivo. This vector may have future utility in murine models of renal disease.

Generation and selection of targeted adenoviruses embodying optimized vector properties.

The utility of adenovirus serotype 5 (Ad5)-based vectors for gene therapy applications would be improved by cell-specific targeting. However, strategies to redirect Ad5 vectors to alternate cellular receptors via replacement of the capsid fiber protein have often resulted in structurally unstable vectors. In view of this, we hypothesized that the selection of modified adenoviruses during their rescue and propagation would be a straightforward approach that guarantees the generation of functional, targeted vectors. Based on our first generation fiber-fibritin molecule, several new chimeric fibers containing variable amounts of fibritin and the Ad5 fiber shaft were analyzed via a new scheme for Ad vector selection. Our selected chimera, composed of the entire Ad5 fiber shaft fused to the 12th coiled-coil segment of fibritin, is capable of efficient capsid incorporation and ligand display. Moreover, transduction by the resultant vector is independent of the expression of the native Ad5 receptor. The incorporation of the Fc-binding domain of Staphylococcus aureus protein A at the carboxy terminus of this chimeric fiber facilitates targeting of the vector to a variety of cellular receptors by means of coupling with monoclonal antibodies. In addition, we have concluded that Ad5 vectors incorporating individual targeting ligands require individual optimization of the fiber-fibritin chimera, which may be accomplished by selecting the optimal fiber-fibritin variant at the stage of rescue of the virus in cells of interest, as described herein.