Transfer of tumor-specific T-cell receptor (TCR) genes into patient T cells is a promising strategy in cancer immunotherapy. We describe here a novel vector (CD8-LV) derived from lentivirus, which delivers genes exclusively and specifically to CD8 ؉ cells. CD8-LV mediated stable in vitro and in vivo reporter gene transfer as well as efficient transfer of genes encoding TCRs recognizing the melanoma antigen tyrosinase. Strikingly, T cells genetically modified with CD8-LV killed melanoma cells reproducibly more efficiently than CD8 ؉ cells transduced with a conventional lentiviral vector. Neither TCR expression levels, nor the rate of activation-induced death of transduced cells differed between both vector types. Instead, CD8-LV transduced cells showed increased granzyme B and perforin levels as well as an up-regulation of CD8 surface expression in a small subpopulation of cells. Thus, a possible mechanism for CD8-LV enhanced tumor cell killing may be based on activation of the effector functions of CD8 ؉ T cells by the vector particle displaying OKT8-derived CD8-scFv and an increase of the surface density of CD8, which functions as coreceptor for tumor-cell recognition. CD8-LV represents a powerful novel vector for TCR gene therapy and other applications in immunotherapy and IntroductionIn the human body, a vast diversity of immune cells constantly patrols the blood stream and tissues to protect from invaders. Each type of these immune cells fulfills different functions. Genetic modification of these cells is a key technology to elucidate their physiologic functions and to develop novel therapeutic strategies. Among the different types of gene vector systems available, ␥-retroviral and lentiviral vectors (LVs) have become state-of-theart technology for lymphocyte gene transfer. [1][2][3] Failure to distinguish between subtypes of cells and thereby transferring genes to both target and nontarget cells is a limitation of vector systems currently in use. Selective and specific delivery of transgenes into particular types of lymphocytes is highly desirable for immunotherapy and gene therapy. Although few attempts have been undertaken to retarget LVs to CD3 ϩ T cells, 4 the transduction of subpopulations or even the transfer of therapeutic genes by such targeting vectors has not been described. In addition, no targeting vector specific for CD8 ϩ T cells has been described. CD8 ϩ T cells are among the most important immune cell types and also a primary target for immunotherapy because of their capacity to directly engage and kill pathogen infected cells or tumor cells. 5 Adoptive transfer of tumor-specific T cells is a promising strategy of directed tumor cell killing, which is currently under investigation in clinical trials worldwide. 6-9 Tumor specificity is provided by an antigen receptor, which can be natural (T-cell receptor; TCR) or engineered (chimeric antigen receptor, CAR). Whereas TCR gene-modified T cells recognize peptide-major histocompatibility complex (pMHC), CAR recognize antigen in an MHC-independen...

Glioma regression requires the recruitment of potent antitumor immune cells into the tumor microenvironment. Dendritic cells (DC) play a role in immune responses to these tumors. The fact that DC vaccines do not effectively combat high-grade gliomas, however, suggests that DCs need to be genetically modified specifically to promote their migration to tumor relevant sites. Previously, we identified extracellular signal-regulated kinase (ERK1) as a regulator of DC immunogenicity and brain autoimmunity. In the current study, we made use of modern magnetic resonance methods to study the role of ERK1 in regulating DC migration and tumor progression in a model of high-grade glioma. We found that ERK1-deficient mice are more resistant to the development of gliomas, and tumor growth in these mice is accompanied by a higher infiltration of leukocytes. ERK1-deficient DCs exhibit an increase in migration that is associated with sustained Cdc42 activation and increased expression of actin-associated cytoskeleton-organizing proteins. We also demonstrated that ERK1 deletion potentiates DC vaccination and provides a survival advantage in high-grade gliomas. Considering the therapeutic significance of these results, we propose ERK1-deleted DC vaccines as an additional means of eradicating resilient tumor cells and preventing tumor recurrence.

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<p>Supplemental Methods</p>

Duchenne muscular dystrophy (DMD) is the most common fatal genetic disease characterized by progressive muscle wasting, loss of ambulation, and typically death in the third decade of life due to respiratory and cardiac complications. DMD results from deleterious mutations in the dystrophin gene that disrupts the translational reading frame and causes a complete lack of dystrophin protein. Becker muscular dystrophy (BMD) is similar to DMD in that it is the result of deletions in the dystrophin gene. However these deletions maintain the translational reading frame and result in the production of an internally truncated but partially functional dystrophin protein. The BMD phenotype is typically less severe than DMD, and thus converting DMD to a BMD-like phenotype by restoring the dystrophin reading frame is a widely explored therapeutic strategy. CRISPR/Cas9 can target precise loci to make specific DNA sequence changes in the genome. We have previously used S. pyogenes Cas9 (SpCas9) to restore dystrophin expression in immortalized myoblasts from DMD patients by deleting dystrophin exon 51 to repair the disrupted reading frame. A promising therapeutic application of this strategy involves in vivo viral delivery of the CRISPR/Cas9 system by AAV, however AAV cannot efficiently package the large SpCas9 gene along with full size promoters. Therefore we have made use of AAV delivery of the smaller S. aureus Cas9 (SaCas9) to delete exon 23 in the mouse dystrophin gene in vivo in the mdx mouse model of DMD and showed restored dystrophin expression and functional recovery. Here we are continuing this work by preparing a SaCas9 system targeted to the human dystrophin gene. gRNAs were designed to target the intronic regions flanking exon 51 and tested in vitro in HEK293T cells as well as immortalized DMD patient myoblasts that are correctable by removal of exon 51. The expected deletion was confirmed by PCR and sequencing of the genomic DNA and dystrophin cDNA. Western blot of lysates from differentiated cells confirmed restoration of dystrophin protein expression. We have also demonstrated exon 51 deletion in vivo following AAV delivery of this CRISPR/Cas9 system to the muscles of a mouse model containing the full length human dystrophin gene. Ongoing work involves testing this approach in a novel dystrophic mouse model carrying a mutated version of the human dystrophin gene that is correctable by exon 51 deletion. This work is important to the continued development of a translational strategy for gene editing as a potentially curative treatment for DMD.

<p>Supplemental Methods</p>