K.K. Wong

Enhanced Long-Term Transduction and Multilineage Engraftment of Human Hematopoietic Stem Cells Transduced with Tyrosine-Modified Recombinant Adeno-Associated Virus Serotype 2

The search for the ideal stem cell gene therapy vector continues as recognized problems persist. Although recombinant adeno-associated virus serotype 2 (rAAV2) mediates gene transfer into hematopoietic stem cells, identified restrictions to transgene expression reduce overall efficiency. Studies have shown that transduction efficiencies are significantly improved by preventing early proteasomal degradation after mutation of surface-exposed tyrosine residues on the capsid to phenylalanine. Here, we report that transduction of human cord blood CD34(+) stem cells by tyrosine-modified rAAV2 is significantly enhanced both in vitro and in vivo. Serial long-term in vivo bioluminescent imaging of immune-deficient recipients after xenotransplantation of CD34(+) cells transduced with tyrosine-modified rAAV2-luciferase revealed that modification of rAAV2 capsids led to a significant increase in the transduction of human CD34(+) cells, without adversely affecting engraftment capacity, or the ability to undergo multilineage differentiation and self-renewal. Together with observations of sustained high-level transgene expression in vivo and efficient persistence of rAAV genomes in human hematopoietic cells, these results suggest that, because of their ability to bypass restrictions to transduction, tyrosine-modified rAAV vectors, particularly Y500F, Y700F, Y444F, and Y704F, represent highly promising candidates for therapeutic evaluation for diseases of human hematopoietic stem cells.

International Patterns of Practice in the Management of Radiation Therapy-induced Nausea and Vomiting

PURPOSEnTo investigate international patterns of practice in the management of radiation therapy-induced nausea and vomiting (RINV).nnnMETHODS AND MATERIALSnOncologists prescribing radiation therapy in the United States, Canada, The Netherlands, Australia, New Zealand, Spain, Italy, France, Hong Kong, Singapore, Cyprus, and Israel completed a Web-based survey that was based on 6 radiation therapy-only clinical cases modeled after the minimal-, low-, moderate-, and high-emetic risk levels defined in the antiemetic guidelines of the American Society of Clinical Oncology and the Multinational Association of Supportive Care in Cancer. For each case, respondents estimated the risks of nausea and vomiting separately and committed to an initial management approach.nnnRESULTSnIn total, 1022 responses were received. Risk estimates and management decisions for the minimal- and high-risk cases varied little and were in line with guideline standards, whereas those for the low- and moderate-risk cases varied greatly. The most common initial management strategies were as follows: rescue therapy for a minimal-risk case (63% of respondents), 2 low-risk cases (56% and 80%), and 1 moderate-risk case (66%); and prophylactic therapy for a second moderate-risk case (75%) and a high-risk case (95%). The serotonin (5-HT)₃ receptor antagonists were the most commonly recommended prophylactic agents. On multivariate analysis, factors predictive of a decision for prophylactic or rescue therapy were risk estimates of nausea and vomiting, awareness of the American Society of Clinical Oncology antiemetic guideline, and European Society for Therapeutic Radiology and Oncology membership.nnnCONCLUSIONSnRisk estimates and management strategies for RINV varied, especially for low- and moderate-risk radiation therapy cases. Radiation therapy-induced nausea and vomiting are under-studied treatment sequelae. New observational and translational studies are needed to allow for individual patient risk assessment and to refine antiemetic guideline management recommendations.

International radiation oncology trainee decision making in the management of radiotherapy-induced nausea and vomiting

PurposeThis study explored international radiation oncology trainee decision making in the management of radiotherapy-induced nausea and vomiting (RINV).MethodsRadiation oncology trainees who were members of the national radiation oncology associations of the USA, Canada, Netherlands, Australia, New Zealand, France, Spain and Singapore completed a Web-based survey. Respondents estimated the risks of nausea and vomiting associated with six standardised radiotherapy-only clinical case vignettes modelled after international anti-emetic guidelines and then committed to prophylactic, rescue or no therapy as an initial management approach for each case.ResultsOne hundred and seventy-six trainees from 11 countries responded. Only 28xa0% were aware of any anti-emetic guideline. In general, risk estimates and management approaches for the high-risk and minimal risk cases varied less and were more in line with guideline standards than were estimates and approaches for the moderate- and low-risk cases. Prophylactic therapy was the most common approach for the high-risk and a moderate-risk case (83 and 71xa0% of respondents respectively), while rescue therapy was the most common approach for a second moderate-risk case (69xa0%), two low-risk cases (69 and 76xa0%) and a minimal risk case (68xa0%). A serotonin receptor antagonist was the most commonly recommended prophylactic agent. On multivariate analysis, a higher estimated risk of nausea predicted for recommending prophylactic therapy, and a lower estimated risk of nausea predicted for recommending rescue therapy.ConclusionsRadiation oncology trainee risk estimates and recommended management approaches for RINV clinical case vignettes varied and matched guideline standards more often for high-risk and minimal risk cases than for moderate- and low-risk cases. Risk estimates of nausea specifically were strong predictors of management decisions.

Clinical oncology and palliative medicine as a combined specialty--a unique model in Hong Kong.

The importance of early integration of palliative care (PC) into oncology treatment is increasingly being recognized. However, there is no consensus on what is the optimal way of integration. This article describes a unique model in Hong Kong where clinical oncology and palliative medicine (PM) is integrated through the development of PM as a subspecialty under clinical oncology.

A Capillary Electrophoresis Sequencing Method for the Identification of Mutations in the Inverted Terminal Repeats of Adeno-Associated Virus

Inverted terminal repeat (ITR) integrity is critical for the replication, packaging, and transduction of recombinant adeno-associated virus (rAAV), a promising gene therapy vector. Because AAV ITRs possess 70% GC content and are palindromic, they are notoriously difficult to sequence. The purpose of this work was to develop a reliable ITR sequencing method. The ITRs of two molecular clones of AAV2, pTZAAV and pAV2, were (1) sequenced directly from plasmid DNA in the presence of denaturant (direct sequencing method, DSM) or (2) first amplified in a reaction in which 7-deaza-dGTP was substituted for dGTP and the resultant amplification product sequenced (amplification sequencing method, ASM). The DSM and ASM techniques yielded clear chromatograms, read through the ITR hairpin, and revealed hitherto unreported mutations in each ITR. pTZAAV and pAV2 possess identical mutations at the upstream MscI site of the 5 ITR (T>G, nt 2) and the downstream MscI site of the 3 ITR (del. nt 4672-4679). The chromatograms for pAV2 also revealed that the ITRs of this construct were arranged in a FLOP/FLOP orientation. In addition, the DSM was successfully employed to recover ITR-chromosomal junction sequences from a variety of rAAV-transduced tissue types. Both the DSM and ASM can be employed to sequence through the AAV ITR hairpin, and both techniques reliably detect mutations in the ITR. Because the DSM and ASM offer a way to verify ITR integrity, they constitute powerful tools for the process development of rAAV gene therapy.

55. AAVHSC Vectors Encoding Zinc-Finger Nucleases Mediate Efficient Targeted Integration at the Human AAVS1 Locus in CD34+ Human Hematopoietic Stem Cells

Zinc-finger nucleases (ZFN) are powerful genome editing tools that may be used to prevent expression of specified genes, correct mutations, or insert transgenes at safe harbor loci. ZFNs create double-stranded breaks at specified locations in the genome which may subsequently be repaired via the non-homologous end joining pathway to interrupt expression of a gene. Targeted integration of genes at pre-determined sites in the genome can be achieved through homology-directed repair of ZFN-mediated targeted double-strand breaks using a co-delivered donor construct. We recently isolated and characterized a panel of novel AAVs (AAVHSC) derived from CD34+ human hematopoietic stem cells (HSC) from healthy donors. We have previously shown that vectors derived from AAVHSCs have the ability to efficiently transduce CD34+ cells. Here we tested the hypothesis that delivery of ZFNs and donor DNA by AAVHSC vectors to CD34+ human cells results in the creation of double stranded site-specific breaks and subsequent efficient targeted transgene integration. A promoterless GFP gene was targeted to Intron 1 of the PPP1R12C gene in the AAVS1 locus such that expression would be driven by the endogenous PPP1R12C promoter following site specific transgene integration. ZFNs specific for PPP1R12C Intron 1 and donor DNA were packaged in AAVHSCs and used to transduce either K562 cells or primary human cytokine-primed peripheral blood CD34+ cells (PBSC). Flow-cytometric analysis at different time points post-transduction revealed specific GFP expression in ZFN + Donor-transduced cells but not in cells transduced with ZFN alone. The multiplicities of infection, ratio of ZFN:donor, and timing of transduction were optimized in K562 cells. Simultaneous transduction of AAVHSC encoding both ZFN and donor DNA at multiplicities of infection of 50,000 and 150,000 respectively was found to be optimal and resulted in GFP expression in up to 50% of transduced cells. Optimized conditions were confirmed in primary CD34+ PBSC from different donors. Targeted integration into AAVS1 was consistently found to be more efficient in CD34+ PBSC as compared with K562 cells, with GFP expression ranging from 10 to 65% of cells. Integrated GFP was expressed long term, with approximately 20% GFP positive cells persisting at 35 days post-transduction in vitro in the absence of selection. Targeted integration into the AAVS1 locus was independently confirmed by amplification using transgene-specific and chromosome-specific primers. Sequencing of the amplified vector chromosome junctions confirmed site-specific integration into AAVS1. No unexpected modifications were found at the junction of the AAVS1 chromosomal sequence and donor homology arms. These results indicate that AAVHSCs function as efficient delivery tools for genome editing in therapeutically relevant cells such as primary PBSC.

96. Genomic Integration Profile of AAVHSC Vectors in Human CD34+ Long-Term Engrafted Hematopoietic Stem Cell Xenografts in Immune-Deficient Mice

Adeno-associated virus vectors (AAV) are proving to be promising and powerful for gene delivery due to their ability to safely transduce both non-dividing and dividing cells. While AAV mainly persists in episomal copies in non-dividing cells, genomic integration has been reported including in CD34+ hematopoietic stem cells (HSCs). Characterizing the vector integration profile is important to ensure safety since genomic integration has the potential for inducing insertional mutagenesis and genotoxicity. The absence of genotoxicity of AAV has been documented in multiple studies. Our lab has recently described the isolation and gene transfer properties of human HSC derived AAVs (AAVHSCs) (Smith LJ, et al. Mol Therapy 2014). To test the ability of the AAVHSCs to transduce long-term engrafted HSCs, we transduced human CD34+ cells with AAV vectors encoding a luciferase transgene prior to transplantation into irradiated immunodeficient NOD/SCID mice. Through serial bioluminescent imaging, we found that the AAVHSCs supported higher levels of expression than AAV2, 7 and 8. Of the AAVHSC serotypes tested, AAVHSC17 was found to support the highest level of transgene expression in vivo, for at least six months post-transplantation. Since vector genomes were found to persist in both long-term engrafted CD34+ cells as well as their differentiated hematopoietic progeny, we hypothesized that the vector genomes likely persisted as chromosomal integrants since episomal copies would have been lost during mitosis. Here, we evaluated the vector genome integration profile in the long-term engrafted CD34+ HSCs transduced with AAVHSC17. High molecular weight DNA was extracted from flow sorted human CD34+ cells isolated from engrafted mouse marrow and sonicated to fragment the DNA before ligation of double stranded DNA adaptors. Using LM-PCR we amplified AAV-chromosomal sequences using AAV-specific and adaptor-specific primers, and further enriched vector-chromosome junctions with nested PCR. The PCR generated library was then used for paired-end Illumina sequencing. The sequence reads were paired using CLC Genomics Workbench. High-throughput analysis resources including PLAN, BLAST (NCBI), and ENSEMBL were used to determine the locations of the AAV-chromosomal junctions. AAV integration was found to occur randomly throughout the genome, including the X and Y chromosomes. Similar to previous studies, AAV integration junctions were largely found in non-coding and intronic regions. Integration was also found to occur within microsatellite repeat regions and GC rich regions. Thus far, no integration events have been found in coding regions or near oncogenes. Due to the lack of detectable genotoxicity resulting from AAV integration in CD34+ HSCs and the ability to support long term in vivo transgene expression, we conclude that these vectors are well-suited for stem cell gene delivery.

‘Who’, ‘when’ and ‘how’ in re-irradiation of recurrent painful bone metastases

Re-irradiation of painful bony metastases is increasingly performed since patients are receiving better systemic treatments and having longer life expectancy, and may also be due to the increase use of initial single fraction radiotherapy. However, randomized control trial on the efficacy of re-irradiation is lacking. A recent meta-analysis concluded with a 58% response rate for pain relief by re-irradiation of symptomatic bone metastases. In this review, the effectiveness of re-irradiation in terms of clinical and economical aspects, and clinical questions on who, when, and how to re-irradiate would be discussed. A brief review of other treatment options and comparison with re-irradiation of bone metastases would be performed.