Delivery and Protection of Adenoviruses Using Biocompatible Hydrogels for Localized Gene Therapy  Rachel Maddox Schek, Scott J Hollister, Paul H Krebsbach  Molecular Therapy  Volume 9, Issue 1, Pages 130-138 (January 2004) DOI: 10.1016/j.ymthe.2003.10.002 Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Expression of β-gal demonstrates infection of cells by virus suspended in gels. Lac-Z virus was incubated in medium with 10% serum for (A) 0, (B) 2, or (C) 24 h or 0.5 mg/ml fibrin gel for (D) 0, (E) 2, or (F) 24 h before being exposed to cells. X-gal staining of these cells allowed visualization of the infected cells. Photographs demonstrate the decrease in number of infected cells as preincubation time was lengthened and that virus contained in fibrin gel was active for longer periods. Molecular Therapy 2004 9, 130-138DOI: (10.1016/j.ymthe.2003.10.002) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 2 The activity of the adenovirus suspended in gel depends on the concentration and composition of gel. Rates of infection were assessed visually using image processing software. (A) Adenovirus was suspended in α-MEM and preincubated before being used to infect cells. Transduction levels declined rapidly with lengthened preincubation times. (B) Virus-containing collagen gels were preincubated with elution fluid; the fluid was removed and cells were seeded on the remaining gels. Infection levels dropped with longer preincubation times and transduction levels were consistently higher for the collagen concentration of 0.5 mg/ml (square) than for 2 mg/ml (circle). (C) Virus contained in fibrin gels exhibited a slower drop in infection rates over lengthened preincubation times. Infection levels were higher for the lower concentration of fibrin, 0.5 mg/ml (square), than for 5 (circle) or 25 mg/ml (triangle). Molecular Therapy 2004 9, 130-138DOI: (10.1016/j.ymthe.2003.10.002) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 3 The concentration and composition of gel from which the virus eluted effects its bioactivity over time. (A) Elution fluid from the collagen gels applied to cells showed that infection levels drop as the preincubation time of the gel increases. Higher numbers of infected cells were observed for all incubation times for the 0.5 mg/ml (square) than for the 2 mg/ml (circle) gel. (B) Elution medium from the fibrin gels was applied to cells. For 5 (circle) and 25 mg/ml (triangle), a drop in transduction levels was observed as incubation times increased, but the 0.5 mg/ml (square) elution fluid showed consistently high transduction levels for up to 48 h of preincubation and measurable activity for more than 60 h. Molecular Therapy 2004 9, 130-138DOI: (10.1016/j.ymthe.2003.10.002) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Elution of viral DNA is much slower and less complete from collagen gel than from fibrin. Real-time PCR was used to quantify the viral DNA present in gels and elution fluids after periods of incubation at 37°C. Standards of known amounts of virus allowed interpolation of the number of viral particles in each sample. (A) Levels of viral particles contained in the collagen gel (white) remained much higher than in the elution medium (black) at all times. (B) In fibrin gel, viral particle levels decreased in the gel (white) and they increased in the elution medium (black). Molecular Therapy 2004 9, 130-138DOI: (10.1016/j.ymthe.2003.10.002) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Fibrin led to significantly more bone formation than either liquid or collagen. After 4 weeks, μCT analysis revealed the presence of bone in 4 of 5 of the fibrin-implanted muscles, 4 of 5 of the collagen-implanted muscles, and 5 of 10 of the liquid-implanted muscles. The volume of the ossicles that were formed using virus in fibrin was significantly larger than that of those formed using either collagen or liquid. No bone was formed in the 1- or 2-week implants. Molecular Therapy 2004 9, 130-138DOI: (10.1016/j.ymthe.2003.10.002) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 6 Both collagen and fibrin gels serve as effective carriers for delivery of functional genes in vivo. Three-dimensional reconstructions of μCT data were used to calculate the volume of the bone formed after 109 viral particles of adenovirus coding for BMP-7 were suspended in culture medium, collagen, or fibrin gels and implanted into the quadriceps of immunocompromised mice. Hematoxylin and eosin staining confirmed (A) the absence of bone in negative control transplants and (B) the presence of ossicles in specimens implanted with the virus; (C) three dimensional rendering of the μCT data. Molecular Therapy 2004 9, 130-138DOI: (10.1016/j.ymthe.2003.10.002) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions