Ribaxamase, an Orally Administered ß-Lactamase, Diminishes Changes to Acquired Antimicrobial Resistance of the Gut Resistome in Patients Treated with Ceftriaxone.

INTRODUCTION: Intravenous (IV) ß-lactam antibiotics, excreted through bile into the gastrointestinal (GI) tract, may disrupt the gut microbiome by eliminating the colonization resistance from beneficial bacteria. This increases the risk for Clostridium difficile infection (CDI) and can promote antimicrobial resistance by selecting resistant organisms and eliminating competition by non-resistant organisms. Ribaxamase is an orally administered ß-lactamase for use with IV ß-lactam antibiotics (penicillins and cephalosporins) and is intended to degrade excess antibiotics in the upper GI before they can disrupt the gut microbiome and alter the resistome. METHODS: Longitudinal fecal samples (349) were collected from patients who participated in a previous Phase 2b clinical study with ribaxamase for prevention of CDI. In that previous study, patients were treated with ceftriaxone for a lower respiratory tract infection and received concurrent ribaxamase or placebo. Extracted fecal DNA from the samples was subjected to whole-genome shotgun sequencing and analyzed for the presence of antimicrobial resistance (AMR) genes by alignment of sequences against the Comprehensive Antibiotic Resistance Database. A qPCR assay was also used to confirm some of the results. RESULTS: Database alignment identified ~1300 acquired AMR genes and gene variants, including those encoding ß-lactamases and vancomycin resistance which were significantly increased in placebo vs ribaxamase-treated patients following antibiotic exposure. qPCR corroborated the presence of these genes and supported both new acquisition and expansion of existing gene pools based on no detectable copy number or a low copy number in pre-antibiotic samples which increased post-antibiotics. Additional statistical analyses demonstrated significant correlations between changes in the gut resistome and clinical study parameters including study drug assignment and ß-lactamase and vancomycin resistance gene frequency. DISCUSSION: These findings demonstrated that ribaxamase reduced changes to the gut resistome subsequent to ceftriaxone administration and may help limit the emergence of AMR.

Neutralization of pertussis toxin by a single antibody prevents clinical pertussis in neonatal baboons.

Pertussis continues to cause considerable infant mortality world-wide, which could be addressed in part by passive immunization strategies. Antibody hu1B7 is a candidate therapeutic that potently neutralizes pertussis toxin in vitro, prevents leukocytosis in mice and treats established disease in weanling baboons as part of an antibody cocktail. Here, we evaluated the potential for hu1B7 and an extended half-life hu1B7 variant to prevent death, leukocytosis and other clinical symptoms in a newborn baboon model that mimics many aspects of human disease. We administered a single antibody dose to newborn baboons five weeks prior to experimental infection. While all animals were heavily colonized with Bordetella pertussis, prophylaxed animals showed significantly greater survival (P < 0.005), delayed and suppressed leukocytosis (P < 0.01) and enhanced clinical outcomes, including coughing (P < 0.01), as compared to controls. Together, this work demonstrates that a single neutralizing anti-PTx antibody is sufficient to prevent clinical pertussis symptoms.

SYN-007, an Orally Administered Beta-Lactamase Enzyme, Protects the Gut Microbiome from Oral Amoxicillin/Clavulanate without Adversely Affecting Antibiotic Systemic Absorption in Dogs.

Beta-lactamases, enzymes produced by bacteria to degrade beta-lactam antibiotics, have been harnessed as therapeutics to protect the gut microbiome from damage caused by antibiotics. Proof-of-concept of this approach using SYN-004 (ribaxamase), a beta-lactamase formulated for oral delivery with intravenous (IV) penicillins and cephalosporins, was demonstrated with animal models and in humans. Ribaxamase degraded ceftriaxone in the gastrointestinal tract, protected the gut microbiome, significantly reduced the incidence of Clostridioides difficile disease and attenuated emergence of antibiotic resistant organisms. SYN-007 is a delayed release formulation of ribaxamase intended for use with oral beta-lactams. In dogs treated with oral amoxicillin, SYN-007 diminished antibiotic-mediated microbiome disruption and reduced the emergence of antibiotic resistance without altering amoxicillin systemic absorption. Here, SYN-007 function in the presence of clavulanate, a beta-lactamase inhibitor, was investigated. Dogs received amoxicillin (40 mg/kg, orally (PO), three times a day (TID)) or the combined antibiotic/beta-lactamase inhibitor, amoxicillin/clavulanate (40 mg/kg amoxicillin, 5.7 mg/kg clavulanate, PO, TID) +/- SYN-007 (10 mg, PO, TID) for five days. Serum amoxicillin levels were not significantly different +/- SYN-007 compared to amoxicillin alone or amoxicillin/clavulanate alone as controls for both first and last doses, indicating SYN-007 did not interfere with systemic absorption of the antibiotic. Whole genome shotgun metagenomics analyses of the fecal microbiomes demonstrated both amoxicillin and amoxicillin/clavulanate significantly reduced diversity and increased the frequency of antibiotic resistance genes. Microbiome damage appeared more severe with amoxicillin/clavulanate. In contrast, with SYN-007, microbiome diversity was not significantly altered, and frequency of antibiotic resistance genes did not increase. Importantly, SYN-007 functioned in the presence of clavulanate to protect the gut microbiome indicating that SYN-007 activity was not inhibited by clavulanate in the dog gastrointestinal tract. SYN-007 has the potential to expand microbiome protection to beta-lactam/beta-lactamase inhibitor combinations delivered orally or systemically.

Oral Beta-Lactamase Protects the Canine Gut Microbiome from Oral Amoxicillin-Mediated Damage.

Antibiotics damage the gut microbiome, which can result in overgrowth of pathogenic microorganisms and emergence of antibiotic resistance. Inactivation of antibiotics in the small intestine represents a novel strategy to protect the colonic microbiota. SYN-004 (ribaxamase) is a beta-lactamase formulated for oral delivery intended to degrade intravenously administered beta-lactam antibiotics in the gastrointestinal (GI) tract. The enteric coating of ribaxamase protects the enzyme from stomach acid and mediates pH-dependent release in the upper small intestine, the site of antibiotic biliary excretion. Clinical benefit was established in animal and human studies in which ribaxamase was shown to degrade ceftriaxone in the GI tract, thereby preserving the gut microbiome, significantly reducing Clostridioides difficile disease, and attenuating antibiotic resistance. To expand ribaxamase utility to oral beta-lactams, delayed release formulations of ribaxamase, SYN-007, were engineered to allow enzyme release in the lower small intestine, distal to the site of oral antibiotic absorption. Based on in vitro dissolution profiles, three SYN-007 formulations were selected for evaluation in a canine model of antibiotic-mediated gut dysbiosis. Dogs received amoxicillin (40 mg/kg, PO, TID) +/- SYN-007 (10 mg, PO, TID) for five days. Serum amoxicillin levels were measured after the first and last antibiotic doses and gut microbiomes were evaluated using whole genome shotgun sequence metagenomics analyses of fecal DNA prior to and after antibiotic treatment. Serum amoxicillin levels did not significantly differ +/- SYN-007 after the first dose for all SYN-007 formulations, while only one SYN-007 formulation did not significantly reduce systemic antibiotic concentrations after the last dose. Gut microbiomes of animals receiving amoxicillin alone displayed significant loss of diversity and emergence of antibiotic resistance genes. In contrast, for animals receiving amoxicillin + SYN-007, microbiome diversities were not altered significantly and the presence of antibiotic resistance genes was reduced. These data demonstrate that SYN-007 diminishes amoxicillin-mediated microbiome disruption and mitigates emergence and propagation of antibiotic resistance genes without interfering with antibiotic systemic absorption. Thus, SYN-007 has the potential to protect the gut microbiome by inactivation of beta-lactam antibiotics when administered by both oral and parenteral routes and to reduce emergence of antibiotic-resistant pathogens.

Oral Metallo-Beta-Lactamase Protects the Gut Microbiome From Carbapenem-Mediated Damage and Reduces Propagation of Antibiotic Resistance in Pigs.

Antibiotics can damage the gut microbiome, leading to serious adventitious infections and emergence of antibiotic resistant pathogens. Antibiotic inactivation in the GI tract represents a strategy to protect colonic microbiota integrity and reduce antibiotic resistance. Clinical utility of this approach was established when SYN-004 (ribaxamase), an orally-administered beta-lactamase, was demonstrated to degrade ceftriaxone in the GI tract and preserve the gut microbiome. Ribaxamase degrades penicillins and cephalosporin beta-lactams, but not carbapenems. To expand this prophylactic approach to include all classes of beta-lactam antibiotics, a novel carbapenemase, formulated for oral administration, SYN-006, was evaluated in a porcine model of antibiotic-mediated gut dysbiosis. Pigs (20 kg, n = 16) were treated with the carbapenem, ertapenem (ERT), (IV, 30 mg/kg, SID) for 4 days and a cohort (n = 8) also received SYN-006 (PO, 50 mg, QID), beginning the day before antibiotic administration. ERT serum levels were not statistically different in ERT and ERT + SYN-006 groups, indicating that SYN-006 did not alter systemic antibiotic levels. Microbiomes were evaluated using whole genome shotgun metagenomics analyses of fecal DNA collected prior to and after antibiotic treatment. ERT caused significant changes to the gut microbiome that were mitigated in the presence of SYN-006. In addition, SYN-006 attenuated emergence of antibiotic resistance, including encoded beta-lactamases and genes conferring resistance to a broad range of antibiotics such as aminoglycosides and macrolides. SYN-006 has the potential to become the first therapy designed to protect the gut microbiome from all classes of beta-lactam antibiotics and reduce emergence of carbapenem-resistant pathogens.

Identification, Characterization, and Formulation of a Novel Carbapenemase Intended to Prevent Antibiotic-Mediated Gut Dysbiosis.

Antibiotics can damage the gut microbiome leading to opportunistic infections and the emergence of antibiotic resistance. Microbiome protection via antibiotic inactivation in the gastrointestinal (GI) tract represents a strategy to limit antibiotic exposure of the colonic microbiota. Proof of concept for this approach was achieved with an orally-administered beta-lactamase enzyme, SYN-004 (ribaxamase), that was demonstrated to degrade ceftriaxone excreted into the GI tract and protect the gut microbiome from antibiotic-mediated dysbiosis. Ribaxamase efficiently degrades penicillin and cephalosporin beta-lactam antibiotics, but is not active against carbapenems. To expand this microbiome protection strategy to include all classes of beta-lactams, three distinct carbapenemases were evaluated for manufacturability, antibiotic degradation spectrum, and stability in human intestinal fluid. E. coli production strains were generated for P2A, a novel metallo-enzyme isolated from B. cereus, New Delhi metallo-beta-lactamase (NDM), and Klebsiella pneumoniae carbapenemase (KPC). While all three enzymes effectively inactivated a broad range of antibiotics, including penicillins, most cephalosporins, and carbapenems in vitro, only P2A retained biological activity when incubated with human chyme. As functional stability in the intestinal tract is a key requirement for an orally-delivered enzyme, P2A was chosen as a potential clinical candidate. An enteric formulation of P2A was developed, called SYN-006, that was inert under high acid conditions, with enzyme dissolution occurring at pH > 5.5. SYN-006 has the potential to expand microbiome protection via antibiotic inactivation to include all classes of beta-lactam antibiotics.

Low dose oral beta-lactamase protects the gut microbiome from oral beta-lactam-mediated damage in dogs.

Antibiotics, while lifesaving, damage the gut microbiome and can precipitate proliferation of pathobionts. A strategy to preserve gut microbiome integrity is to eliminate biologically active antimicrobials excreted into the gastrointestinal tract (GI) without negatively affecting antibiotic therapeutic efficacy. Clinical proof of concept was achieved with SYN-004 (ribaxamase), a beta-lactamase enzyme formulated for oral delivery with intravenous penicillins and cephalosporins. Ribaxamase inactivated intestinal ceftriaxone, protected the gut microbiome, and significantly reduced the incidence of Clostridioides difficile disease. For use with oral beta-lactam antibiotics, a delayed release formulation of ribaxamase, SYN-007, was engineered for dissolution in the lower small intestine distal to the site of oral antibiotic absorption. In dogs that received oral amoxicillin, SYN-007 reduced microbiome disruption without interfering with amoxicillin systemic absorption. Here, a study to determine the lowest effective dose of SYN-007 was performed. Dogs received amoxicillin (40 mg/kg, PO, TID) +/- SYN-007 (PO, TID) at three doses, 10 mg, 3 mg, or 1 mg for five days. Serum amoxicillin levels, measured after the first and last antibiotic doses, were not significantly different +/-SYN-007 at all dose levels indicating that SYN-007 did not interfere with amoxicillin systemic absorption. Microbiome analyses demonstrated that amoxicillin significantly reduced bacteria richness and microbiome diversity resulting in altered microbiome composition. However, with all doses of SYN-007, microbiome richness and diversity were not significantly different from pretreatment and changes in microbiome composition were attenuated. These data demonstrate that effective SYN-007 doses can be reduced at least 10-fold while maintaining gut microbiome preservation. The potential to employ low SYN-007 doses to protect the gut microbiota has important implications for enhancing therapeutic outcomes for patients receiving oral beta-lactam antibiotics while simultaneously reducing cost per dose and ultimately, healthcare expenses.

Distinct consequences of amoxicillin and ertapenem exposure in the porcine gut microbiome.

The gut microbiome influences many, if not all, aspects of human health. Antibiotics, while lifesaving, have the unintended consequence of killing commensal microbiota inhabiting the gastrointestinal (GI) tract, which can lead to overgrowth of opportunistic pathogens such as Clostridium difficile and emergence of antibiotic-resistant organisms. Here, porcine models were developed to evaluate changes to the gut microbiome caused by two distinct types of beta-lactam antibiotics delivered via common administration routes, oral amoxicillin and intravenous ertapenem. Amoxicillin is one of the most often used broad-spectrum antibiotics, frequently prescribed to young children. Ertapenem, a carbapenem considered a last resort antibiotic, is used sparingly in humans and prohibited for use in animals. Cohorts of normal pigs (n = 5) were treated with amoxicillin (20 mg/kg, PO, BID) or ertapenem (30 mg/kg, IV, SID) for seven days. Microbiomes were evaluated using whole genome shotgun metagenomics analyses of fecal DNA collected prior to, during, and after antibiotic treatment. Each antibiotic resulted in significant and distinct changes in the microbiome, causing elimination of key commensal bacterial species and overgrowth of other, potentially pathogenic taxa. In addition, amoxicillin promoted propagation of a broad range of antibiotic resistance genes, many encoding efflux pump components and beta-lactamases, while ertapenem triggered emergence of genes encoding vancomycin resistance, and beta-lactamases, including the carbapenemase, IMP-27. Notably, microbiota alterations and antibiotic resistance gene propagation displayed unique patterns following exposure to amoxicillin or ertapenem. These data underscore the importance of understanding consequences of individual antibiotic use to predict and potentially mitigate adverse outcomes. The porcine models developed here can facilitate evaluation of therapeutic interventions to prevent antibiotic-mediated microbiome disruption.

Prior exposure to Bordetella species as an exclusion criterion in the baboon model of pertussis.

The baboon model of Bordetella pertussis infection is the newest and most clinically accurate model of the human disease to date. However, among the 15 experimentally infected baboons in this study, a subset of baboons did not exhibit the expected high bacterial colonization levels or increase in white blood cell count. Moreover, cultures of nasopharyngeal wash samples from several baboons suggested B. bronchiseptica coinfection. Analysis of serum antibodies recognizing filamentous hemagglutinin, pertussis toxin and B. pertussis lipo-oligosaccharide indicated that several baboons had likely been previously exposed to Bordetella species and that prior exposure correlated with partial protection from B. pertussis infection. Notably, all animals with a baseline Fha titer of 5 IU/ml or below exhibited symptoms typical of the model, suggesting this value can be used as inclusion criteria for animals prior to study enrollment. While B. pertussis infection is endemic to human populations and B. bronchiseptica is common in wild small mammals, this study illustrates that baboons can readily harbor both organisms. Awareness of Bordetella species that share antigens capable of generating protective immune responses and tracking of prior exposure to those species is required for successful use of the baboon model of pertussis.

Development of SYN-004, an oral beta-lactamase treatment to protect the gut microbiome from antibiotic-mediated damage and prevent Clostridium difficile infection.

The gut microbiome, composed of the microflora that inhabit the gastrointestinal tract and their genomes, make up a complex ecosystem that can be disrupted by antibiotic use. The ensuing dysbiosis is conducive to the emergence of opportunistic pathogens such as Clostridium difficile. A novel approach to protect the microbiome from antibiotic-mediated dysbiosis is the use of beta-lactamase enzymes to degrade residual antibiotics in the gastrointestinal tract before the microflora are harmed. Here we present the preclinical development and early clinical studies of the beta-lactamase enzymes, P3A, currently referred to as SYN-004, and its precursor, P1A. Both P1A and SYN-004 were designed as orally-delivered, non-systemically available therapeutics for use with intravenous beta-lactam antibiotics. SYN-004 was engineered from P1A, a beta-lactamase isolated from Bacillus licheniformis, to broaden its antibiotic degradation profile. SYN-004 efficiently hydrolyses penicillins and cephalosporins, the most widely used IV beta-lactam antibiotics. In animal studies, SYN-004 degraded ceftriaxone in the GI tract of dogs and protected the microbiome of pigs from ceftriaxone-induced changes. Phase I clinical studies demonstrated SYN-004 safety and tolerability. Phase 2 studies are in progress to assess the utility of SYN-004 for the prevention of antibiotic-associated diarrhea and Clostridium difficile disease.

Steroid and anticonvulsant prophylaxis for stereotactic radiosurgery: Large variation in physician recommendations.

PURPOSE/OBJECTIVE(S): The risk of developing symptomatic edema or seizure following stereotactic radiosurgery (SRS) is poorly defined, and many practitioners prescribe prophylactic corticosteroids and/or anticonvulsants. Because there are no clear guidelines regarding appropriate use, we sought to characterize prescribing practices and factors associated with these recommendations. METHODS AND MATERIALS: We conducted a 1-time, internet-based survey among 500 randomly selected radiation oncologists self-described as specializing in central nervous system diseases who were registered in the American Society for Radiation Oncology directory. Physicians were contacted by e-mail and invited to complete the 22-question survey. RESULTS: The response rate was 32% (n = 161). Sixty-six percent of respondents had been in practice for >10 years, and 45% of respondents practiced at an academic medical center. During/after SRS, 53% of respondents "always" or "usually" recommended corticosteroids, whereas 47% "never," "rarely," or "sometimes" recommended them. When prescribing corticosteroids, the recommended duration of use was <1 week, 1-2 weeks, or >2 weeks among 49%, 33%, and 18% of respondents, respectively. Respondents who worked in an academic medical center were less likely to prescribe corticosteroids, although this did not reach significance (P = .09). Seizure prophylaxis was less common overall, as 79% of respondents "rarely" or "never" prescribed anticonvulsants for SRS. Respondents who prescribed anticonvulsants more frequently had higher estimations of the risk of seizure within 2 weeks of SRS (P < .001), and their recommended duration of anticonvulsant use was <1 week, 1-2 weeks, and >2 weeks among 35%, 25%, and 41% of respondents, respectively. CONCLUSIONS: There is extreme variation in physician recommendations regarding prophylactic corticosteroid and anticonvulsant use for patients undergoing SRS. Further investigation of the risks and benefits of these medications for SRS is warranted, which may promote guideline development and more patient-centered, rational prescribing practices.

Sustained delivery of a HIF-1 antagonist for ocular neovascularization.

Doxorubicin (DXR) and daunorubicin (DNR) inhibit hypoxia-inducible factor-1 (HIF-1) transcriptional activity by blocking its binding to DNA. Intraocular injections of DXR or DNR suppressed choroidal and retinal neovascularization (NV), but also perturbed retinal function as demonstrated by electroretinograms (ERGs). DXR was conjugated to novel copolymers of branched polyethylene glycol and poly(sebacic acid) (DXR-PSA-PEG3) and formulated into nanoparticles that when placed in aqueous buffer, slowly released small DXR-conjugates. Intraocular injection of DXR-PSA-PEG3 nanoparticles (1 or 10 µg DXR content) reduced HIF-1-responsive gene products, strongly suppressed choroidal and retinal NV, and did not cause retinal toxicity. In transgenic mice that express VEGF in photoreceptors, intraocular injection of DXR-PSA-PEG3 nanoparticles (10 µg DXR content) suppressed NV for at least 35 days. Intraocular injection of DXR-PSA-PEG3 nanoparticles (2.7 mg DXR content) in rabbits resulted in sustained DXR-conjugate release with detectable levels in aqueous humor and vitreous for at least 105 days. This study demonstrates a novel HIF-1-inhibitor-polymer conjugate formulated into controlled-release particles that maximizes efficacy and duration of activity, minimizes toxicity, and provides a promising new chemical entity for treatment of ocular NV.

Day-case Karydakis flap for pilonidal sinus.

PURPOSE: The Karydakis flap for pilonidal sinus is associated with primary wound healing and infrequent recurrence. Previous studies had reported in-patient protocols. This cohort study was designed to assess the feasibility, safety, and practicalities of day-case Karydakis flap surgery. Factors relating to wound healing also were explored. METHODS: Consecutive patients undergoing day-case Karydakis flap surgery, by one consultant surgeon, for pilonidal sinus were studied prospectively. Patients were assessed at weekly intervals after surgery until healing was complete. Wound healing time was compared with 1) patients' gender, age, body mass index, deprivation index, occupation and smoking status, 2) pilonidal diseases' dimensions and proximity to anus, 3) wounds' dimensions and proximity to anus, and 4) drain volume. RESULTS: Day-case Karydakis flap surgery was feasible, safe, and effective. None of the 51 patients in the study required readmission to hospital, sepsis drainage, or surgery for recurrent sinus. Median wound healing time was three weeks. Smokers healed quicker than nonsmokers. No other factors were identified that were associated with delayed healing. Normal activity was resumed within one month of surgery in 95 percent of patients. CONCLUSIONS: The Karydakis flap can offer the advantage of day-case surgery for pilonidal sinus patients in addition to primary wound healing and low sinus recurrence rates.

Complete sequence and organization of the human adenovirus serotype 46 genome.

Out of 51 human adenoviral serotypes recognized to date, 32 of them belong to species D. Members of species D adenoviruses are commonly isolated from immune suppressed patients (organ transplant) and patients suffering from AIDS. The role of species D adenoviruses in pathogenesis is currently unclear. To derive new insights into the genetic content and evolution of species D adenoviruses and as a first step towards development of human adenovirus serotype 46 (Ad46) as vector, the complete nucleotide sequence of the virus was determined. The size of the genome is 35,178 bp in length with a G+C content of 56.9%. All the early and late region genes are present in the expected locations of the genome. The deduced amino acid sequences of all late region genes, with the exception of fiber, exhibited high degree of homology with the corresponding proteins of other adenoviruses. The deduced amino acid sequences of early regions E1, E3 and E4 showed a high degree of homology with the corresponding proteins of adenoviruses belonging to species D and less homology with the corresponding proteins of adenoviruses of other species. The homologues of Ad5 E3 region genes encoding 12.5K, gp19K, 10.4K, 14.5K and 14.7K are conserved in the genome of Ad46. However, the E3 region of Ad46 lacks genes encoding 6.7K and adenovirus death protein (ADP) but contains two additional open reading frames with a coding capacity of 433 and 281 amino acids. The fiber protein of Ad46 is 200 amino acids smaller than the fiber protein of Ad5 and contains only 10 pseudo-repeats in the shaft region. To facilitate the manipulation of the genome, the complete genome of Ad46 was cloned into a single bacterial plasmid. Following transfection into E1 complementing cell lines, the virus was recovered demonstrating the feasibility of viral genome manipulation for generation of recombinant viruses.

Increased expression of VEGF in retinal pigmented epithelial cells is not sufficient to cause choroidal neovascularization.

Increased expression of vascular endothelial cell growth factor (VEGF) in the retina is sufficient to stimulate sprouting of neovascularization from the deep capillary bed of the retina, but not the superficial retinal capillaries or the choriocapillaris. Coexpression of VEGF and angiopoietin 2 (Ang2) results in sprouting of neovascularization from superficial and deep retinal capillaries, but not the choriocapillaris. However, retina-derived VEGF and Ang2 may not reach the choriocapillaris, because of tight junctions between retinal pigmented epithelial (RPE) cells. To eliminate this possible confounding factor, we used the human vitelliform macular dystrophy 2 (VMD2) promoter, an RPE-specific promoter, combined with the tetracycline-inducible promoter system, to generate double transgenic mice with inducible expression of VEGF in RPE cells. Adult mice with increased expression of VEGF in RPE cells had normal retinas and choroids with no choroidal neovascularization (CNV), but when increased expression of VEGF in RPE cells was combined with subretinal injection of a gutless adenoviral vector containing an expression construct for Ang2 (AGVAng2), CNV consistently occurred. In contrast, triple transgenic mice with induced expression of Ang2 and VEGF in RPE cells, did not develop CNV. These data suggest that increased expression of VEGF and/or Ang2 in RPE cells is not sufficient to cause CNV unless it is combined with a subretinal injection of a gutless adenoviral vector, which is likely to perturb RPE cells. These data also suggest that the effects of angiogenic proteins may vary among vascular beds, even those that are closely related, and, therefore, generalizations should be avoided.

Intraocular gutless adenoviral-vectored VEGF stimulates anterior segment but not retinal neovascularization.

Vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) have been implicated as important stimulatory factors for retinal neovascularization. In this study, we used intraocular gene transfer with gutless adenoviral (AGV) vectors to determine the effect of increased intraocular expression of VEGF, IGF-1, or sphingosine kinase (SPK), which produces sphingosine-1-phosphate, another angiogenic factor. Retinal neovascularization did not occur from intravitreous AGV-vectored VEGF, IGF-1, SPK, or combined VEGF and IGF-1, except occasionally adjacent to the retinal penetration site from the injection. However, corneal and iris neovascularization occurred after 2 weeks in all eyes injected with AGV.VEGF, but not those injected with only AGV.IGF-1 or AGV.SPK. These data suggest that the superficial capillary bed of the retina is relatively insensitive to VEGF, IGF-1, or SPK in adult mice, except when combined with retinal trauma. However, AGV-vectored VEGF is sufficient to consistently cause severe corneal and iris neovascularization. This provides a model for anterior segment neovascularization, which unlike previous models is relatively inexpensive and is not plagued by spontaneous regression, and therefore, may be useful for identification of new treatments.

Angiopoietin-2 enhances retinal vessel sensitivity to vascular endothelial growth factor.

Increased expression of vascular endothelial growth factor (VEGF) in the retina starting after postnatal day (P)7 results in neovascularization originating from deep retinal capillaries, but not those in the superficial capillary bed. Doxycycline was administered starting P0 to double transgenic mice with inducible expression of VEGF in the retina. These mice showed proliferation and dilation of superficial retinal capillaries, indicating that at this stage of development, the superficial capillaries are sensitive to the effects of VEGF. Angiopoietin-2 (Ang2) is expressed along the surface of the retina for several days after birth, but by P7 and later, Ang2 is only expressed in the region of the deep capillary bed. In mice with ubiquitous doxycycline-inducible expression of Ang2, in the absence of doxycycline, intravitreous injection of a gutless adenoviral vector expressing VEGF (AGV.VEGF) resulted in neovascularization of the cornea and iris, but no retinal neovascularization. After treatment with doxycycline to induce Ang2 expression, intravitreous injection of AGV.VEGF caused retinal neovascularization in addition to corneal and iris neovascularization. The retinal neovascularization originated from both the superficial and deep capillary beds. These data suggest that Ang2 promotes sensitivity to the angiogenic effects of VEGF in retinal vessels.

Delivery of adenoviral DNA to mouse liver.

The liver represents a major target organ for gene delivery owing to its high biosynthetic capacity and access to the bloodstream. Adenoviral vectors are highly efficient gene-transfer vehicles, making them among the most promising systems for in vivo gene transfer to the liver. Following intravenous administration of adenoviral vectors to a variety of mammalian models, including mice, dogs, and monkeys, hepatocytes are efficiently transduced. Several delivery methods to the liver have been described, including portal vein (2-4), hepatic artery (3,5), and peripheral vein infusions (6). This chapter describes the simple, nonsurgical method of intravenous (iv) administration of adenoviral vectors in mice, and an immunohistochemical method to qualitatively evaluate liver transduction efficiency following delivery of an adenoviral vector encoding a bgalactosidase (beta-gal) marker gene. Additionally, several alternative methods to verify efficient liver transduction are introduced.

Potentiation of oncolytic adenoviral vector efficacy with gutless vectors encoding GMCSF or TRAIL.

Oncolytic adenoviral vectors selectively replicate in and lyse human tumor cells, providing a promising means for targeted tumor destruction. However, oncolytic vectors have limited capacity for incorporation of additional genetic material that could encode therapeutic transgenes and/or transcriptional regulatory control elements to augment the efficacy and/or safety of the vector. Therefore, we hypothesized that coadministration of an oncolytic vector with a replication-defective, gutless adenoviral vector encoding a therapeutic transgene would result in replication of both vectors within a tumor and potentiate antitumor efficacy relative to the use of either vector alone. We constructed gutless vectors encoding the murine granulocyte-macrophage colony-stimulating factor (AGVmGMF) or human tumor necrosis factor alpha-related apoptosis-inducing ligand (AGVhTRAIL) gene and tested the ability of these vectors to augment the efficacy of an oncolytic vector (Ar6pAE2fE3F) in a potentiating vector strategy. In Hep3B cells in vitro, cotreatment with Ar6pAE2fE3F increased transgene expression from AGVhTRAIL and permitted replication of AGVhTRAIL, suggesting that an oncolytic vector can propagate gutless vector spread in vivo. In pre-established Hep3B xenograft tumors, neither gutless vector alone inhibited tumor growth; however, coadministration of AGVmGMF or AGVhTRAIL with Ar6pAE2fE3F significantly reduced tumor growth relative to Ar6pAE2fE3F alone. Additionally, use of AGVhTRAIL with Ar6pAE2fE3F increased the number of complete or partial tumor regressions observed at study end. These data provide evidence that coadministration of an oncolytic vector with a gutless vector holds promise for potentiating tumor ablation efficacy.

Development of adenovirus serotype 35 as a gene transfer vector.

While 51 human adenoviral serotypes have been identified to date, the vast majority of adenoviral vectors designed for gene transfer have been generated in the adenovirus serotype 5 (Ad5) backbone. Viral infections caused by Ad5 are endemic in most human populations and the majority of humans carry preexisting humoral and/or cellular immunity to Ad5 which may severely limit the use of Ad5-based vectors for gene therapy applications. To circumvent this preexisting Ad5 immunity, we have identified Ad35 as an alternative adenoviral serotype to which the majority of humans do not have neutralizing antibodies. Importantly, Ad35 can be grown to high titers with a low particle-to-PFU ratio. As a prerequisite for the development of Ad35 for use as a gene transfer vector, a genome organization map was constructed using the available Ad35 sequence information, and E1a-deficient Ad35 vectors encoding marker genes were generated. Ad35 biodistribution in mice was assessed following intravenous administration and compared with that of Ad5. Extremely low levels of Ad35 were detected in all organs evaluated, including liver, lung, spleen, and bone marrow, while Ad5 displayed high transduction of these organs. Due to the lack of Ad35 liver tropism, minimal hepatotoxicity was observed in mice treated with Ad35. Furthermore, the half-life of Ad35 in mouse blood was found to be two to three times longer than that of Ad5. These data suggest that either mice do not express the Ad35 cell surface receptor or that Ad35 does not efficiently transduce mouse cells in vivo following systemic delivery. Therefore, to begin to elucidate the Ad35 cell entry mechanisms, in vitro competition studies were performed. These data demonstrated that Ad35 cell entry is CAR independent, and may involve protein(s) expressed on most human cells.