Gene Therapy for the Enhancement of Fracture Healing - Engineered Bone: Tissue Engineering Intelligence Unit - page 133

In humans, two phase 1 clinical trials for gene therapy of cystic fibrosis have been under-
taken. 67,68 AAV is of relatively small size. Therefore, all viral genes are deleted in the production
of AAV vectors for the treatment of cystic fibrosis. This means that the least amount of immu-
nogenic material remains. However, this intervention leaves little space for an optimal pro-
moter sequence. Site specificity of the integration is lost with the deletion of the AAV rep gene,
required for replication. However, it has been shown that even with the rep deletion, AAV
vectors have been capable of long-term persistence through random-site integration and episo-
mal persistence. 69
Preliminary results of a not yet completed dose-escalation study including 19 subjects with
mild cystic fibrosis lung disease have shown that administration of AAV vectors does not in-
duce inflammatory responses over a wide range of doses. 67
In a second study, administration of AAV vectors to the paranasal sinuses of cystic fibrosis
patients resulted in gene transfer to the epithelium at a rate of one copy of the gene for every 10
cells. This level of transfer appeared to be stable for up to 10 weeks. 68
Kay et al 70 reported on a clinical trial of gene therapy in severe hemophilia B. Based on
preclinical studies demonstrating efficacy and absence of vector-related toxicity in hemophilic
mice and dog models, an AAV vector encoding for human F IX was injected intramuscular in
adults. Results regarding safety, gene transfer and expression in the first three patients were
encouraging and gave no evidence of toxicity, transfer in semen or formation of inhibitory
antibodies against F IX.
Orthopedic Gene Therapy with Recombinant Adeno-Associated Virus Vectors
An inflammatory arthritis rodent model was used to evaluate the efficacy of the recombi-
nant AAV vectors for orthopedics therapy. Two studies using the same model described their
findings; Pan et al 71 reported 95% of inflamed synoviocytes expressed the beta-galactosidase
driven by a cytomegalovirus promoter (AAV-CMV-LacZ). Goater et al 72 also showed transgene
expression of beta-galactosidase in arthritic knees. The data indicated a 10-fold increase in the
reporter gene expression in the arthritic knee compared to the control. The preliminary work
with AAV vectors for gene therapy is encouraging and we anticipate a larger number of future
studies especially since methods for producing a high titer 54 and purification 56 have regularly
been published. These advances will allow further studies using AAV vectors. In general, AAV
vectors hold promise for gene therapy as they appear to be safe and have superior duration
profiles. 73,74
Nonviral Gene Therapy
The limitations of viral vectors, in particular their relatively small capacity for therapeutic
DNA, safety concerns, (such as insertional mutagenesis and toxicity problems), and difficulty
in targeting to specific cell types have led to the evaluation and development of alternative
vectors based on chemically nonviral systems. Advantages of nonviral methods of DNA trans-
fer include their capability of transporting large size DNA inserts, their safety regarding mu-
tagenic issues, and their lesser immunogenicity. Furthermore, using pharmaceutical techniques
large scale production should be possible. Disadvantages of non viral vectors comprise their
poor transfection efficiencies, which in part is due to endolysosomal degradation 75 and only
transient expression due to the lack of integration in the host genome. Although, in certain
circumstances a transient expression is desired.
To develop successful non viral vectors, researchers have to understand and optimize the
mechanisms involved in the traffic of DNA to its target destination. The three barriers that the
DNA has to overcome are: (1) The DNA has to be transported to the cytoplasm, either directly
through the plasma membrane or by escape from the endosome; (2) The DNA has to be
translocated to the nucleus; or (3) For many applications the DNA has either to be integrated
into the genome or extrachromosomally replicated. Many approaches were investigated to effi-
ciently transition the DNA through these steps. Below we will discuss the different approaches
to transfer non viral DNA.