Drug delivery in acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia was among the first malignancies to be cured by drug therapy alone, but overall survival rates remain unsatisfactory and have changed little over the past 20 years. Conventional chemotherapeutic regimens, which almost invariably include cytarabine and anthracyclines, are untargeted, and more specific therapies are needed. OBJECTIVE: We have chosen acute myeloid leukemia as a disease prototype to review established and novel targeted approaches in leukemia treatment. METHODS: Our selection of the reviewed literature focused on drug delivery aspects. CONCLUSION: While the toxicity profile of chemotherapeutics has been improved by liposomal formulations and antibody conjugation for leukemia-directed uptake, their efficacy has probably not changed significantly. Drugs with an alternative mode of action, including kinase inhibitors, hold great promise. Further improvements may result from the characterization of novel acute myeloid leukemia (AML) cell surface receptors and of leukemic stem cells, as well as from the design of leukemia-targeted gene therapy vectors.

Novel mutations in the CLN6 gene causing a variant late infantile neuronal ceroid lipofuscinosis.

The neuronal ceroid lipofuscinoses (NCLs) are a heterogeneous group of autosomal recessive neurodegenerative diseases comprising Batten and other related diseases plus numerous variants. They are characterized by progressive neuronal cell death. The CLN6 gene was recently identified, mutations in which cause one of the variant late infantile forms of NCL (vLINCL). We describe four novel mutations in the CLN6 gene. This brings the total number of CLN6 mutations known to 11 in 38 families. This suggests that the CLN6 gene may be highly mutable. An American patient of Irish/French/Native American origin was heterozygous for a 4-bp insertion (c.267_268insAACG) in exon 3. The other allele had a point mutation (c.898T>C) in exon 7 resulting in a W300R amino acid change. Two Trinidadian siblings of Indian origin were homozygous for a mutation at the 5' donor splice site of exon 4 (IVS4+1G>T), affecting the first base of the invariant GT at the beginning of intron 4. The fourth novel mutation, a double deletion of 4 bp and 1 bp in exon 7 (c.829_832delGTCG;c.837delG), was identified in a Portuguese patient heterozygous for the I154del Portuguese CLN6 mutation. Four of the 11 mutations identified are in exon 4. Three Portuguese patients with clinical profiles similar to CLN6 patients without defects in CLN6 or other known NCL genes are described. We conclude the following: 1) the CLN6 gene may be a highly mutable gene; 2) exon 4 must code for a segment of the protein crucial for function; 3) vLINCL disease in Portugal is genetically heterogeneous; 4) the I154del accounts for 81.25% of affected CLN6 Portuguese alleles; and 5) three vLINCL Portuguese patients may have defects in a new NCL gene.

Novel cytotoxic vectors based on adeno-associated virus.

Vectors based on adeno-associated virus (AAV) are promising tools for gene therapy. The production of strongly toxic vectors, for example for cancer-directed gene transfer, is often unfeasible due to uncontrolled expression of toxic genes in vector-producing cells. Using an approach based on transcriptional repression, we have created novel AAV vectors carrying the genes coding for diphtheria toxin A (DTA) and the pro-apoptotic PUMA protein. The DTA vector had a significant toxic effect on a panel of tumor cell lines, and abrogation of protein synthesis could be shown. The PUMA vector had a toxic effect on HeLa and RPMI 8226 cells, and sensitized transduced cells to doxorubicin. To permit targeted gene transfer, we incorporated the DTA gene into a genetically modified AAV-2 capsid previously developed by our group that mediates enhanced transduction of murine breast cancer cells in vitro. This vector had a stronger cytotoxic effect on breast cancer cells than DTA vectors with wildtype AAV capsid or vectors with a random capsid modification. The vector production and application system presented here allows for easy exchange of promotors, transgenes and capsid specificity for certain target cells. It will therefore be of great possible value in a broad range of applications in cytotoxic gene therapy and significantly broadens the spectrum of available tools for AAV-based gene therapy.

Successful expansion but not complete restriction of tropism of adeno-associated virus by in vivo biopanning of random virus display peptide libraries.

Targeting viral vectors to certain tissues in vivo has been a major challenge in gene therapy. Cell type-directed vector capsids can be selected from random peptide libraries displayed on viral capsids in vitro but so far this system could not easily be translated to in vivo applications. Using a novel, PCR-based amplification protocol for peptide libraries displayed on adeno-associated virus (AAV), we selected vectors for optimized transduction of primary tumor cells in vitro. However, these vectors were not suitable for transduction of the same target cells under in vivo conditions. We therefore performed selections of AAV peptide libraries in vivo in living animals after intravenous administration using tumor and lung tissue as prototype targets. Analysis of peptide sequences of AAV clones after several rounds of selection yielded distinct sequence motifs for both tissues. The selected clones indeed conferred gene expression in the target tissue while gene expression was undetectable in animals injected with control vectors. However, all of the vectors selected for tumor transduction also transduced heart tissue and the vectors selected for lung transduction also transduced a number of other tissues, particularly and invariably the heart. This suggests that modification of the heparin binding motif by target-binding peptide insertion is necessary but not sufficient to achieve tissue-specific transgene expression. While the approach presented here does not yield vectors whose expression is confined to one target tissue, it is a useful tool for in vivo tissue transduction when expression in tissues other than the primary target is uncritical.