[show abstract][hide abstract] ABSTRACT: Alpha-1 antitrypsin (AAT) is a multi-functional protein that has anti-inflammatory and tissue protective properties. We previously reported that human AAT (hAAT) gene therapy prevented autoimmune diabetes in non-obese diabetic (NOD) mice and suppressed arthritis development in combination with doxycycline in mice. In the present study we investigated the feasibility of hAAT monotherapy for the treatment of chronic arthritis in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA).
DBA/1 mice were immunized with bovine type II collagen (bCII) to induce arthritis. These mice were pretreated either with hAAT protein or with recombinant adeno-associated virus vector expressing hAAT (rAAV-hAAT). Control groups received saline injections. Arthritis development was evaluated by prevalence of arthritis and arthritic index. Serum levels of B-cell activating factor of the TNF-α family (BAFF), antibodies against both bovine (bCII) and mouse collagen II (mCII) were tested by ELISA.
Human AAT protein therapy as well as recombinant adeno-associated virus (rAAV8)-mediated hAAT gene therapy significantly delayed onset and ameliorated disease development of arthritis in CIA mouse model. Importantly, hAAT therapies significantly reduced serum levels of BAFF and autoantibodies against bCII and mCII, suggesting that the effects are mediated via B-cells, at least partially.
These results present a new drug for arthritis therapy. Human AAT protein and gene therapies are able to ameliorate and delay arthritis development and reduce autoimmunity, indicating promising potential of these therapies as a new treatment strategy for RA.
Journal of Translational Medicine 02/2011; 9:21. · 3.46 Impact Factor
[show abstract][hide abstract] ABSTRACT: DNA-dependent protein kinase (DNA-PK) is a DNA repair enzyme and plays an important role in determining the molecular fate of the rAAV genome. However, the effect this cellular enzyme on rAAV DNA replication remains elusive.
In the present study, we characterized the roles of DNA-PK on recombinant adeno-associated virus DNA replication. Inhibition of DNA-PK by a DNA-PK inhibitor or siRNA targeting DNA-PKcs significantly decreased replication of AAV in MO59K and 293 cells. Southern blot analysis showed that replicated rAAV DNA formed head-to-head or tail-to-tail junctions. The head-to-tail junction was low or undetectable suggesting AAV-ITR self-priming is the major mechanism for rAAV DNA replication. In an in vitro replication assay, anti-Ku80 antibody strongly inhibited rAAV replication, while anti-Ku70 antibody moderately decreased rAAV replication. Similarly, when Ku heterodimer (Ku70/80) was depleted, less replicated rAAV DNA were detected. Finally, we showed that AAV-ITRs directly interacted with Ku proteins.
Collectively, our results showed that that DNA-PK enhances rAAV replication through the interaction of Ku proteins and AAV-ITRs.
PLoS ONE 01/2010; 5(12):e15073. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Rheumatoid arthritis (RA) is a complex disease characterized by autoimmune inflammation and joint destruction. Despite recent advances in RA treatment, current therapies require further improvement to overcome adverse events and ineffectiveness in some cases. By targeting different pathways/molecules using drug combinations, a better treatment can be obtained, whereas adverse events are reduced. In order to develop a new treatment option, the present study employs a gene therapy-based combination therapy using doxycycline and human alpha-1 antitrypsin (hAAT).
DBA/1 mice were immunized with type II collagen to induce arthritis. Four weeks before immunization, they received a doxycycline containing diet and a single injection of adeno-associated virus vector expressing hAAT under the control of a tetracycline-dependent promoter. Control groups received doxycycline alone or saline. Macroscopic arthritis development as well as histopathological changes in the joint were evaluated. In addition, the effects of hAAT and doxycycline on lipopolysaccharide (LPS)- or tumor necrosis factor-alpha-induced interleukin (IL)-6 production from mouse fibroblast cells were also determined.
Combination therapy significantly reduced arthritis development and progression compared to the control group in respect to macroscopic as well as histopathological changes. Doxycycline and hAAT in combination also inhibited IL-6 expression from LPS-stimulated NIH/3T3 mouse fibroblast cells, indicating a contributing mechanism of arthritis inhibition.
The results obtained in the present study indicate that a combination therapy using AAT and doxycycline holds promising potential as a new therapy for RA.
The Journal of Gene Medicine 10/2009; 12(1):35-44. · 2.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: Alpha 1 antitrypsin (AAT) is a serine proteinase inhibitor (serpin). One well-known function of this protein is to inactivate neutrophil elastase and other neutrophil-derived proteinases, and prevent the destruction of pulmonary extracellular matrix. Deficiency of AAT can cause emphysema due to degradation of interstitial elastin by elastase. The majority of circulating AAT is secreted from the liver. Muscle-directed gene therapy using recombinant adeno-associated virus 2 (rAAV2) vectors has been tested to increase the serum levels of AAT. However, inefficient transduction of rAAV2 vector makes it difficult to reach therapeutic levels of AAT in clinical trials and it remains unclear as to whether muscle-secreted AAT is functional. In the present study, we evaluated five serotypes (1, 2, 3, 4, and 5) of rAAV vectors for transduction efficiency in mouse muscle. Results from these studies showed that rAAV1 is the most efficient vector among these serotypes and mediated at least 100-fold higher levels of AAT secretion than the rAAV2 vector. Western blot analysis showed that this murine muscle-secreted human AAT (hAAT) formed a complex with human neutrophil elastase in a dose-dependent manner. An anti-elastase activity assay showed that murine muscle-secreted hAAT inhibited elastase with equal capacity as hAAT purified from plasma. These results provide strong support for the functionality of AAT in ongoing clinical studies of muscle-directed AAT gene therapy.
The Journal of Gene Medicine 07/2006; 8(6):730-5. · 2.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: alpha1-Antitrypsin (AAT) deficiency is a single-gene disorder in which a mutation in the AAT (approved symbol SERPINA1) gene (PI*Z) leads to misfolding of the protein, loss of the protective antiprotease effect of AAT for the lungs, and a toxic effect on hepatocytes. Optimal therapy for AAT deficiency will require a high percentage of hepatocyte transduction to be effective for liver and lung disease. Recently, rAAV genomes pseudotyped with capsids from serotypes 7 and 8 showed efficient hepatic transduction. We hypothesized that upon portal vein injection to target hepatocytes, serotype 8 would better transduce target cells and therefore express hAAT in both a greater percentage of cells and greater amounts. AAV2 and pseudotyped vectors for serotypes 1, 5, and 8 carrying the human AAT transgene were injected at 1 x 10(10) particle doses into C57Bl/6 mice. Circulating hAAT from AAV2/8-injected animals showed a 2-log advantage over AAV2 and 3-log increase over AAV2/1 and 5 for the 24-week study. Most significantly, up to 40% of total liver cells stained positive for the transgene in AAV2/8 subjects while remaining primarily episomal. Therefore, pseudotyped AAV8 provides a vehicle to infect a high percentage of hepatocytes stably and thereby express therapeutic molecules to modify AAT PiZ transcripts.
[show abstract][hide abstract] ABSTRACT: DNA dependent protein kinase (DNA-PK) is a DNA repair enzyme with multiple functions. It has been shown that it plays an important role in determining the molecular fate of the rAAV genome in mouse muscle and liver. DNA-PKcs inhibits AAV integration in vitro and in vivo. In the present study, we sought to determine the effect of DNA-PKcs on recombinant adeno-associated virus (rAAV) replication. We co-infected 293 cells with rAAV vector (UF5) and recombinant herpes simplex virus (rHSV) helper virus. The replicated forms were detected by southern blot analysis. When cells were treated with a DNA-PKcs inhibitor, wortmannin (WM), rAAV replication is significantly reduced in a dose-dependent manner. Similar results were obtained from MO59K (DNA-PKcs positive) cells. In order to confirm this observation, we have employed small interference RNA (siRNA) to target DNA-PKcs. Transfection of this synthetic siRNA resulted in 70% to 90% reduction of the mRNA and the protein levels of DNA-PKcs. Remarkably, this treatment significantly decreased rAAV replication. In order to avoid the possible side effects by viral infection on DNA-PK activity, we co-transfected the siRNA treated 293 cells with vector (pUF5) and helper (pDG) plasmids. Results from these experiments again showed that targeting of DNA-PKcs decreased rAAV replication by at least two folds compared to the controls. Our results demonstrated that inhibition of DNA-PKcs by a DNA-PK inhibitor or siRNA decreased rAAV replication suggesting the important role of this cellular enzyme in AAV replication.
[show abstract][hide abstract] ABSTRACT: Previously, we showed that 40-50 % vector DNA and transgene expression remained in the SCID (DNA-PKcs deficient) mouse liver after a partial hepatectomy, while less than 10% vector DNA and transgene expression remained in C57BL/6 (B6) mouse liver. These results demonstrate that DNA-PK inhibits rAAV DNA integration. However, it is not clear whether the integration occurred before or after partial hepatectomy and whether cell division affected rAAV integration. In the present study, we sought to test the hypothesis that cell division may enhance rAAV DNA integration. Immediately after a partial hepatectomy, B6 (n=5) or SCID mice (n=3) were intraportally injected with a rAAV vector (rAAV2-CB-AAT, 3×109 i.u./mouse). As a control, the same vector (3×109 i.u./mouse) was also intraportally injected into B6 (n=3) or SCID (n=3) liver without a partial hepatectomy. Transgene expressions in all four groups were evaluated weekly by monitoring serum levels of hAAT. Serum hAAT levels in both hepatectomized SCID and B6 mice were sustained 3 weeks after injection. Six to eight weeks after injection, hAAT levels from hepatectomized SCID liver (dividing model) were nearly 100% of that from intact SCID liver (non dividing model), while hAAT levels from hepatectomized B6 liver (dividing model) were 25-30% of that from intact B6 liver (non-dividing model). Southern blot analyses showed that total copies of the vector DNA in dividing and non-dividing SCID models were comparable, while the episomal forms of the vector DNA were significantly lower in the dividing SCID model than in the non-dividing SCID model. These results strongly suggested that hepatocyte division (in partial-hepatectomized liver) not only reduced episomal forms of rAAV DNA but also enhanced rAAV DNA integration.
[show abstract][hide abstract] ABSTRACT: Allogeneic stem cell-based transplants may be limited by allograft rejection, as is seen with conventional organ transplantation. One way to avert such a response is to use autologous stem cells, but that may carry the risk of recurrence of the original disease, particularly in the context of a genetic defect. We investigated the potential for gene modification of autologous stem cells to avoid both problems, using recombinant adenoassociated virus vector expressing human alpha1-antitrypsin in murine liver progenitor cells. We showed that recombinant adenoassociated virus 1 was the most efficient vector for liver progenitor cell transduction among five different serotypes of recombinant adenoassociated virus vectors. Ex vivo infected green fluorescent protein-positive liver progenitor cells from C57BL/6 mice with recombinant adenoassociated virus 1-vector-expressing human alpha1 antitrypsin were transplanted into the liver of monocrotaline-treated and partial-hepatectomized C57BL/6 recipients. Using green fluorescent protein as a donor marker, we were able to determine that at 18 weeks after transplantation, approximately 40% to 50% of the regenerated liver was green fluorescent protein positive. In addition, transgene expression (serum human alpha1-antitrypsin) was sustained for the length of the study (18 weeks after transplantation). Immunostaining revealed approximately 5% to 10% of repopulating liver cells expressing human alpha1-antitrypsin. In conclusion, this study demonstrated the feasibility of long-term engraftment and stability of transgene expression from genetically modified liver progenitor cells with a recombinant adenoassociated virus vector and implies a novel approach to gene therapy for treatment of liver diseases, such as alpha1-antitrypsin deficiency.
[show abstract][hide abstract] ABSTRACT: Molecular Therapy (2004) 9, S164–S164; doi: 10.1016/j.ymthe.2004.06.384
429. Alpha 1 Antitrypsin Gene Delivery for Preventing Type 1 Diabetes in NOD Mice*
Yuanqing Lu1, Mei Tang1, Clive Wasserfall1, Young-Kook Choi1, Thomas Gardmann1, Martha Campbell-Thompson1, Mark A. Atkinson1,* and Sihong Song1,*1Departments of Pharmaceutics and Pathology, Powell Gene Therapy Center, University of Florida, Gainesville, FL*Sihong Song, Mark A. Atkinson.*This work was supported by grants from the Juvenile Diabetes Research Foundation, NIDDK (DK 62652) and The Alpha One Foundation.
[show abstract][hide abstract] ABSTRACT: Recent studies have shown that recombinant adeno-associated virus (rAAV) can persist in episomal form; however, factors affecting rAAV persistence are poorly understood. DNA-dependent PK (DNA-PK) is a DNA repair enzyme, which we previously found played an important role in determining the molecular fate of the rAAV genome in mouse skeletal muscle. In the present study, we tested the effect of DNA-PK on AAV serotype 2 integration in vitro and in vivo in mouse liver. In an in vitro integration system, addition of DNA-PK decreased AAV integration, whereas antibody against DNA-PKcs increased integration. In vivo, matched doses of a recombinant AAV serotype 2 vector were injected into the portal vein of either C57BL/6 (DNA-PKcs(+/+)) or severe combined immunodeficient (DNA-PKcs(-/-)) mice. After partial hepatectomy to stimulate hepatocyte proliferation, retention of vector genomes and of transgene expression was substantially higher in severe combined immunodeficient mice, indicating that in the absence of DNA-PKcs, a greater proportion of genomes integrated into the cellular genome. In summary, we have provided evidence that DNA-PK inhibits AAV integration both in vitro and in vivo.
Proceedings of the National Academy of Sciences 03/2004; 101(7):2112-6. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: It has been observed that DNA-dependent protein kinase (DNA-PK) inhibits adeno-associated virus (AAV) DNA integration in vitro and in vivo (Song et al PNAS 2004). In order to test the effect of DNA-PK on AAV replication, we have infect MO59J (DNA-PK negative) and MO59K (DNA-PK positive) cells with a recombinant virus (UF5) in the presence or absence of a helper virus (carrying AAV rep and cap genes). Hirt DNA were isolated 2 days after infection, and subjected to southern blot analysis. Data showed that the abundance of replicated monomer of the vector DNA was lower in MO59K cells than in MO59J suggesting DNA-PK may inhibit AAV replication. To avoid the effect from the difference other than DNA-PK between the two cell lines, we have employed the small interfering RNA (siRNA) technology to decrease DNA-PK expression in MO59K cells. Synthetic siRNAs for human DNA-PKcs were transfected into MO59K cells. Total RNA were extracted at 2 or 4 days after transfection. RT-PCR analysis showed that DNA-PKcs mRNA was nearly undetectable after 25, and 35 cycles. These results indicate that siRNAs can efficiently target DNA-PKcs, thus provide a powerful tool for creating cell or animal models to study the cellular effects on AAV replication and integration.