Vision Loss after Intravitreal Injection of Autologous "Stem Cells" for AMD.

Adipose tissue-derived "stem cells" have been increasingly used by "stem-cell clinics" in the United States and elsewhere to treat a variety of disorders. We evaluated three patients in whom severe bilateral visual loss developed after they received intravitreal injections of autologous adipose tissue-derived "stem cells" at one such clinic in the United States. In these three patients, the last documented visual acuity on the Snellen eye chart before the injection ranged from 20/30 to 20/200. The patients' severe visual loss after the injection was associated with ocular hypertension, hemorrhagic retinopathy, vitreous hemorrhage, combined traction and rhegmatogenous retinal detachment, or lens dislocation. After 1 year, the patients' visual acuity ranged from 20/200 to no light perception.

Selection of chronic lymphocytic leukemia binding peptides.

To provide cell-binding ligands for ex vivo gene therapy and chronic lymphocytic leukemia (CLL)-targeting ligands for in vivo drug and gene therapy, we selected 44 20-mer peptides from peptide-presenting phage libraries by panning against primary patient CLL cancer cells. Twenty-nine of the selected peptides were assayed for cell binding. Eight of the selected peptides bound CLL cells, B cells, T cells, and monocyte cells, 12 bound only CLL cells and B cells, and 1 peptide bound only B cells. However, eight of the selected peptides were CLL specific. When two of the peptides were tested out of the context of phage, the synthetic peptides were able to bind cells and functionally retarget adenovirus to increase ex vivo gene delivery to primary CLL cells. These data demonstrate the ability to identify lead cancer-targeting peptides by selection of phage libraries against primary human cancers cells.

Biotinylated gene therapy vectors.

The avidin-biotin system is a fundamental technology in biomedicine for immunolocalisation, imaging, nucleic acid blotting and protein labelling. This technology has recently been adapted for use in gene therapy vector applications to add proteins or cell-targeting ligands to non-viral and viral vectors. Two biotinylation technologies are being used in these applications: chemical biotinylation and metabolic biotinylation. In chemical biotinylation, reactive alkylating agents couple biotin to proteins by random covalent attachment to amino acid side chains. In metabolic biotinylation, proteins are genetically engineered with a biotin acceptor peptide (BAP), such that they are covalently biotinylated by cellular biotin ligases during viral vector production. Both technologies show promise for cell-targeting in vitro and in vivo, and for ligand screening applications. Metabolic biotinylation has the added feature of allowing viruses, vectors and vaccines to be produced from cells already biotinylated, thereby allowing them to purified by affinity chromatography on monomeric avidin columns.

Early osteoblastic differentiation induced by dexamethasone enhances adenoviral gene delivery to marrow stromal cells.

We investigated the implications of induced osteogenic differentiation on gene delivery in multipotent rat marrow stromal cells (MSCs). Prior to genetic manipulation cells were cultured with or without osteogenic supplements (5x10(-8) M dexamethasone, 160 microM l-ascorbic acid 2-phosphate, and 10 mM beta-glycerophosphate). Comparison of liposome, retroviral, and adenoviral vectors demonstrated that all three vectors could mediate gene delivery to primary rat MSCs. When these vectors were applied in the absence or presence of osteogenic supplements, we found that MSCs differentiated prior to transduction with adenovirus type 5 vectors produced a 300% increase in transgene expression compared to MSCs that were not exposed to osteogenic supplements. This differentiation effect appeared specific to adenoviral mediated gene delivery, since there was minimal increase in retroviral gene delivery and no increase in liposome gene delivery when MSCs were treated with osteogenic supplements. In addition, we also determined this increase in transgene production to occur at a higher concentration of dexamethasone (5x10(-8) M) in the culture medium of MSCs prior to adenoviral transduction. We found that this increased transgene production could be extended to the osteogenic protein, human bone morphogenetic protein 2 (hBMP-2). When delivered by an adenoviral vector, hBMP-2 transgene production could be increased from 1.4 ng/10(5) cells/3 days to 4.3 ng/10(5) cells/3 days by culture of MSCs with osteogenic supplements prior to transduction. These results indicate that the utility of MSCs as a therapeutic protein delivery mechanism through genetic manipulation can be enhanced by pre-culture of these cells with dexamethasone.

Nosocomial acute-onset postoperative endophthalmitis at a university teaching hospital (2002-2009).

PURPOSE: To evaluate acute-onset postoperative endophthalmitis occurring at an academic medical center and to compare rates over the last 25 years at a single institution. DESIGN: Retrospective, consecutive case series. METHODS: Medical records were reviewed for all patients diagnosed with acute-onset postoperative nosocomial endophthalmitis from 2002 through 2009 associated with surgery at Bascom Palmer Eye Institute. RESULTS: The 8-year frequency of acute-onset postoperative endophthalmitis was 0.025% (14 of 56 672 intraocular surgeries). The rate was 0.028% (8/28 568) for cataract surgery and 0.011% (2/18 492) for pars plana vitrectomy (PPV). Both PPV endophthalmitis cases followed 20-gauge surgery and no cases followed small-gauge, transconjunctival PPV (n = 2262). Three cases occurred following penetrating keratoplasty (3/2788, 0.108%). The most common bacterial isolate was Staphylococcus (n = 7, 50%). Initial treatment involved ocular paracentesis (n = 8, 57%) or vitrectomy (n = 5, 36%), in combination with injection of intraocular antibiotics (n = 14, 100%). Vancomycin and ceftazidime were used in 13 eyes (93%) and intraocular steroids were given initially to 9 eyes (64%). Final visual acuity was > or =20/200 in 9 eyes (64%) and 2 eyes (14%) were no light perception. At this institution since 1984, there has been a statistically significant trend for a decreasing rate of acute-onset postoperative endophthalmitis (1984-1994: 0.09%; 1995-2001: 0.05%; 2002-2009: 0.025%; P < .001). CONCLUSION: At a university teaching hospital involving resident, fellow, and faculty surgeons, the frequency of acute-onset postoperative nosocomial endophthalmitis is low, has not increased in the era of sutureless clear corneal cataract surgery, and has steadily decreased when compared to prior time periods from the same institution.

Avidin-based targeting and purification of a protein IX-modified, metabolically biotinylated adenoviral vector.

While genetic modification of adenoviral vectors can produce vectors with modified tropism, incorporation of targeting peptides/proteins into the structural context of the virion can also result in destruction of ligand targeting or virion integrity. To combat this problem, we have developed a versatile targeting system using metabolically biotinylated adenoviral vectors bearing biotinylated fiber proteins. These vectors have been demonstrated to be useful as a platform for avidin-based ligand screening and vector targeting by conjugating biotinylated ligands to the virus using high-affinity tetrameric avidin (K(d) = 10(-15) M). The biotinylated vector could also be purified by biotin-reversible binding on monomeric avidin (K(d) = 10(-7) M). In this report, a second metabolically biotinylated adenovirus vector, Ad-IX-BAP, has been engineered by fusing a biotin acceptor peptide (BAP) to the C-terminus of the adenovirus pIX protein. This biotinylated vector displays twice as many biotins and was markedly superior for single-step affinity purification on monomeric avidin resin. However, unlike the fiber-biotinylated vector, Ad-IX-BAP failed to retarget to cells with biotinylated antibodies including anti-CD71 against the transferrin receptor. In contrast, Ad-IX-BAP was retargeted if transferrin, the cognate ligand for CD71, was used as a ligand rather than the anti-CD71. This work demonstrates the utility of metabolic biotinylation as a molecular screening tool to assess the utility of different viral capsid proteins for ligand display and the biology and compatibility of different ligands and receptors for vector targeting applications. These results also demonstrate the utility of the pIX-biotinylated vector as a platform for gentle single-step affinity purification of adenoviral vectors.

Metabolically biotinylated adenovirus for cell targeting, ligand screening, and vector purification.

Development of cell-targeting vectors is an important focus for gene therapy. While some ligands can be genetically inserted into virus capsid proteins for cell targeting, for many ligands, this approach can disrupt either ligand function or vector function. To address this problem for adenovirus type 5 vectors, the fiber capsid protein was genetically fused to a biotin acceptor peptide (BAP). Adenovirus particles bearing this BAP were metabolically biotinylated during vector production by the endogenous biotin ligase in 293 cells to produce covalently biotinylated virions. The resulting biotinylated vector could be retargeted to new receptors by conjugation to biotinylated antibodies using tetrameric avidin (K(d) = 10(-15) M). The biotinylated vector could also be purified by biotin-reversible binding on monomeric avidin (K(d) = 10(-7) M). Finally, this vector was used as a ligand screening platform for dendritic cells in which a variety of structurally diverse protein, carbohydrate, and nucleic acid ligands were easily added to the vector using the biotin-avidin interaction. This work demonstrates the utility of metabolically biotinylated viruses for ligand screening, vector targeting, and virus purification applications.