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Ribozymes 257

absence of any viral gene expression reduces the chance of As the use of ribozymes progresses from cell culture
an immune response in animals. In addition, retroviruses systems to animal models, additional control over ri-
can be easily pseudo-typed with a variety of envelope pro- bozyme expression will be required. Ribozyme expression
teins to broaden or restrict host cell tropism, thus adding can be restricted to specific organs or cell types through
an additional level of cellular targeting for ribozyme gene the use of tissue-specific promoters. This has been done
delivery. Adenoviral vectors can be produced at high successfully in tissue culture using the tyrosinase pro-
titers and provide very efficient transduction, but they moter, which is exclusively expressed in melanocytes. In
do not integrate into the host genome and, consequently, another example, transgenic mice were created that car-
expression of the transgenes is only transient in actively ried a ribozyme gene driven by the insulin promoter, only
dividing cells. Other viral delivery systems are actively expressed in the pancreatic beta-cell islets. Alternatively,
being pursued, such as the adeno-associated virus, alpha- inducible promoters, such as those regulated by tetracy-
viruses, and lentiviruses. Adeno-associated virus is attrac- cline, or steroid hormones, have shown utility both in cell
tive as a small, nonpathogenic virus that can stably in- culture and in animals, allowing ribozyme expression to
tegrate into the host genome. An alphavirus system, be turned on and off at will.
using recombinant Semliki Forest virus, provides high
transduction efficiencies of mammalian cells along with
C. Ribozyme Applications
cytoplasmic ribozyme expression.
Ribozymes have been applied as antiviral agents, treat-
ments for cancer and genetic disorders, and as tools for
B. Ribozyme Gene Expression
pathway elucidation and target validation. Ribozymes are
A number of viral promoters (e.g., cytomegalovirus, unique in that they can inactivate specific gene expression,
retroviruses) utilizing RNA pol II have yielded high and thus can be used to identify the function of a protein or
intracellular expression levels of ribozymes. Furthermore, the role of a gene in a functional cascade. This application,
selectable markers such as antibiotic resistance or cell called target validation, is critical for both basic biological
surface proteins can be coexpressed with the ribozyme research and drug development. Compared to other means
to allow monitoring of ribozyme expression. Embedding of target validation (e.g., transgenic or knockout animals),
ribozymes in a long mRNA transcript can potentially ribozymes offer specificity and ease of design and usage.
have a negative impact on ribozyme localization and Initial uses of ribozymes focused on antivirals, primar-
even folding. One possible solution for this problem is ily for the treatment of HIV. Viruses that go through a ge-
to engineer self-cleaving ribozymes on either side of the nomic RNA intermediate in their replication cycle, such as
functional ribozyme so that it can be released from the HIV, hepatitis B virus, and hepatitis C virus, are attractive
nascent transcript. targets because a single species of ribozyme can target
RNA pol III promoters, including tRNA, adenovirus both viral genomic RNA and mRNAs. Ribozymes have
VA1, U1, and U6 small nuclear RNA derivatives, have also been widely used to target cellular genes, including
allowed ubiquitous and very high intracellular levels of those aberrantly expressed in cancers. One early ribozyme
ribozyme expression. Several creative permutations, such target was the bcr–abl fusion transcript created from the
as the combination of two promoters driving the same ri- Philadelphia chromosome associated with chronic myel-
bozyme gene or the placement of the ribozyme expres- ogenous leukemia. This chromosome is characterized by a
sion cassette within the U3 region of a Long terminal translocation that results in the expression of a transform-
repeat, to produce a “double-copy” retroviral vector ing bcr–abl fusion protein. In this case, ribozymes have
led to further increases in ribozyme expression. Fi- been designed to specifically target the fusion mRNA and
nally, multiple ribozymes can be expressed within the not the normal bcr or abl mRNAs. Ribozymes have also
same RNA transcript or from multiple promoter-ribozyme been designed to specifically target the mutant ras gene
cassettes. while sparing the normal homolog. Ribozymes target-
Intracellular localization of the ribozyme transcript ing overexpressed HER-2/neu in breast carcinoma cells
is another important parameter. Specific strategies to effectively reduced the tumorigenicity of these cells in
colocalize ribozymes with their target RNA have been mice.
developed to maximize intracellular ribozyme activ- In addition to directly targeting oncogenes, ribozymes
ity. These strategies take advantage of the RNA se- have also been applied more indirectly as anticancer ther-
quences flanking the ribozyme. Certain RNA sequences apies. For example, ribozymes targeting the multiple drug
direct specific subcellular localization, making it possi- resistance-1 or fos mRNAs in cancer cell lines effectively
ble to tightly colocalize ribozyme transcripts with their made the cells more sensitive to chemotherapeutic ag-
target RNAs. ents. Alternatively, a ribozyme targeting bcl-2 triggered