Gene Therapy for the Enhancement of Fracture Healing - Engineered Bone: Tissue Engineering Intelligence Unit - page 130

Retroviral vectors have specific advantages for ex vivo gene therapy, and in fact retroviruses
are unfavorable for in vivo approaches. By ex vivo methods, cells can be isolated and propa-
gated in culture, genetically modified by retroviral transduction and finally transplanted into a
recipient. 4,37 Cited studies demonstrate feasibility of retrovirus mediated ex vivo approaches to
bone and fracture healing. The use of progenitor or stem cells, which must be harvested and
isolated prior to delivery, opens exciting avenues for ex vivo gene therapy approaches. Further
studies are required to determine the ideal vector systems that work best for each individual
application. Understanding the workable mechanisms for gene expression regulation will have
major impacts on the future of gene therapy. Research must focus on the basic biology of the
mechanisms of action for target gene and the vector systems used to deliver the genes to cells.
In contrast to retroviral vectors, there is much more literature describing the successes of aden-
oviral gene therapy strategies for orthopaedic applications.
Adenoviral Vectors
Adenoviruses
Adenoviruses are a class of double stranded, linear DNA viruses of approximately 35 kilobases
in length (reviewed by ref. 38). There are more than 40 serotypes of adenoviruses with sero-
types 5 and 2 (Ad.5 and Ad.2) being the most utilized as vectors for gene transfer. The advan-
tages to adenoviral vectors are that they can infect a wide variety of both dividing and nondi-
viding cells, resulting in high, transient levels of gene expression. Thus, adenoviral vectors are
well suited for in vivo gene transfer where short-term gene expression is required. The disad-
vantages to adenoviral vectors are that the vectors are toxic to some cell types at a high multi-
plicity of infection and can stimulate an immune response to the infected cells.
Adenoviruses infect cells through an interaction of the viral fiber protein with its receptor,
CAR. An interaction between a RGD protein motif in the penton base of the virus with a
specific integrin then allows for fusion of the adenovirus to the cell and entry of the linear viral
DNA into cells. Expression of the E1 region of the virus, containing the E1A and E1B open
reading frames, from the adenoviral promoter results in subsequent E1A dependent expression
of the E2, E3 and E4 open reading frames. Finally, following expression of the early genes
important for DNA replication, expression of the viral late genes allows for packaging of the
DNA into the viral capsid and release from the cell by lysis.
Adenovirus Vectors
Given that the temporal order of viral gene expression requires E1A for subsequent expres-
sion of the viral genes needed for viral DNA replication and packaging, deletion of the E1
region results in a replication defective virus. However, the replication defective ∆ E1 virus can
be propagated by gene complementation of E1A and E1B in trans. In particular, the human
embryonic kidney cell line 293, stably transfected with E1A and E1B, allows for propagation
of the E1 deficient viruses. Thus first generation Ad.2 or Ad.5 adenoviral vectors are always
deficient in E1, with the therapeutic or marker transgenes usually inserted into the E1 region
of the virus, under the regulation of a heterologous promoter. In addition, many of the vectors
are deficient in E3 since proteins expressed from differentially spliced E3 transcripts are in-
volved in blocking the immune response to virally infected cells as well as inhibiting apoptosis.
Deletion of the E3 region, in conjunction with deletion of the E1 region results in a larger
capacity of the vector for therapeutic genes. These first generation vectors have been used
extensively for both preclinical studies as well as in clinical trials. However, following infection
with the E1 and E3 deleted vectors, there is still a low level of expression of the E2 and E4
regions of the virus, resulting in toxicity at high viral doses and the induction of an immune
response to the virally infected cells.
In order to reduce the toxicity and immunogenicity of the virus following infection, expres-
sion from the E4 region has been eliminated in second generation vectors. The E4 region
encodes for 7 different open reading frames with Orf3 appearing to be the most important for