Christian J. Buchholz

The Secreted β-Amyloid Precursor Protein Ectodomain APPsα Is Sufficient to Rescue the Anatomical, Behavioral, and Electrophysiological Abnormalities of APP-Deficient Mice

It is well established that the proteolytic processing of the β-amyloid precursor protein (APP) generates β-amyloid (Aβ), which plays a central role in the pathogenesis of Alzheimers disease (AD). In contrast, the physiological role of APP and of its numerous proteolytic fragments and the question of whether a loss of these functions contributes to AD are still unknown. To address this question, we replaced the endogenous APP locus by gene-targeted alleles and generated two lines of knock-in mice that exclusively express APP deletion variants corresponding either to the secreted APP ectodomain (APPsα) or to a C-terminal (CT) truncation lacking the YENPTY interaction motif (APPΔCT15). Interestingly, the ΔCT15 deletion resulted in reduced turnover of holoAPP, increased cell surface expression, and strongly reduced Aβ levels in brain, likely because of reduced processing in the endocytic pathway. Most importantly, we demonstrate that in both APP knock-in lines the expression of APP N-terminal domains either grossly attenuated or completely rescued the prominent deficits of APP knock-out mice, such as reductions in brain and body weight, grip strength deficits, alterations in circadian locomotor activity, exploratory activity, and the impairment in spatial learning and long-term potentiation. Together, our data suggest that the APP C terminus is dispensable and that APPsα is sufficient to mediate the physiological functions of APP assessed by these tests.

Targeted Cell Entry of Lentiviral Vectors

Retargeting of lentiviral vector entry to cell types of interest is a key factor in improving the safety and efficacy of gene transfer. In this study we show that the retargetable envelope glycoproteins of measles virus (MV), namely, the hemagglutinin (H) responsible for receptor recognition and the fusion protein (F), can pseudotype human immunodeficiency virus 1 (HIV-1) vectors when their cytoplasmic tails are truncated. We then pseudotyped HIV-1 vectors with MV glycoproteins displaying on H either the epidermal growth factor or a single-chain antibody directed against CD20, but without the ability to recognize their native receptors. Gene transfer into cells that expressed the targeted receptor was several orders of magnitude more efficient than into cells that did not. High-target versus nontarget cell discrimination was demonstrated in mixed cell populations, where the targeting vector selectively eliminated CD20-positive cells after suicide gene transfer. Remarkably, primary human CD20-positive B lymphocytes were transduced more efficiently by the CD20-targeted vector than by a vector pseudotyped with the vesicular stomatitis virus G (VSV-G) protein. In addition, the CD20-targeted vector was able to transduce even unstimulated primary B cells, whereas VSV-G pseudotyped vectors were unable to do so. Because MV enters cells through direct fusion at the cell membrane, this novel targeting system should be widely applicable.

Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors

We present a flexible and highly specific targeting method for lentiviral vectors based on single-chain antibodies recognizing cell-surface antigens. We generated lentiviral vectors specific for human CD105+ endothelial cells, human CD133+ hematopoietic progenitors and mouse GluA-expressing neurons. Lentiviral vectors specific for CD105 or for CD20 transduced their target cells as efficiently as VSV-G pseudotyped vectors but discriminated between endothelial cells and lymphocytes in mixed cultures. CD133-targeted vectors transduced CD133+ cultured hematopoietic progenitor cells more efficiently than VSV-G pseudotyped vectors, resulting in stable long-term transduction. Lentiviral vectors targeted to the glutamate receptor subunits GluA2 and GluA4 exhibited more than 94% specificity for neurons in cerebellar cultures and when injected into the adult mouse brain. We observed neuron-specific gene modification upon transfer of the Cre recombinase gene into the hippocampus of reporter mice. This approach allowed targeted gene transfer to many cell types of interest with an unprecedented degree of specificity.

Mapping of the primary binding site of measles virus to its receptor CD46

The measles virus (MV) hemagglutinin binds to the complement control protein (CCP) CD46 primarily through the two external modules, CCP-I and -II. To define the residues involved in binding, 40 amino acids predicted to be solvent-exposed on the CCP-I-II module surface were changed to either alanine or serine. Altered proteins were expressed on the cell surface, and their abilities to bind purified MV particles, a soluble form of hemagglutinin (sH) and nine CD46-specific antibodies competing to different levels with sH attachment, were measured. All proteins retained, at least in part, MV and sH binding, but some completely lost binding to certain antibodies. Amino acids essential for binding of antibodies weakly or moderately competing with sH attachment are situated in the membrane-distal tip of CCP-I, whereas residues involved in binding of strongly sH competing antibodies cluster in the center of CCP-I (Arg-25, Asp-27) or in CCP-II (Arg-69, Asp-70). Both clusters face the same side of CCP-I-II and map close to amino acid exchanges impairing sH binding (E11A, R29A, P39A, and D70A) or MV binding (D70A and E84A) and to a six-amino acid loop, previously shown to be necessary for sH binding.

Oncolytic Efficacy and Enhanced Safety of Measles Virus Activated by Tumor-Secreted Matrix Metalloproteinases

Cancer cells secrete matrix metalloproteinases (MMP) that degrade the extracellular matrix and are responsible for some hallmarks of malignant cancer. Many viruses, including a few currently used in oncolytic virotherapy clinical trials, depend on intracellular proteases to process their proteins and activate their particles. We show here for measles virus (MV) that particle activation can be made dependent of proteases secreted by cancer cells. The MV depends on the intracellular protease furin to process and activate its envelope fusion (F) protein. To make F protein activation cancer cell specific, we introduced hexameric sequences recognized by an MMP and identified the mutant proteins most effective in fusing MMP-expressing human fibrosarcoma cells (HT1080). We showed that an MMP inhibitor interferes with syncytia formation elicited by mutant F proteins and confirmed MMP-dependent cleavage by Edman degradation sequence analysis. We generated recombinant MVs expressing the modified F proteins in place of furin-activated F. These viruses spread only in cells secreting MMP. In nude mice, an MMP-activated MV retarded HT1080 xenograft growth as efficiently as the furin-activated MV vaccine strain. In MV-susceptible mice, the furin-activated virus caused lethal encephalitis upon intracerebral inoculation, whereas the MMP-activated did not. Thus, MV particle activation can be made dependent of proteases secreted by cancer cells, enhancing safety. This study opens the perspective of combining targeting at the particle activation, receptor recognition, and selective replication levels to improve the therapeutic index of MV and other viruses in ongoing clinical trials of oncolysis.

DARPins: An Efficient Targeting Domain for Lentiviral Vectors

We have recently developed a retargeting system for lentiviral vectors (LVs) that relies on the pseudotyping of LVs with engineered measles virus (MV) glycoproteins (hemagglutinin (H) and fusion protein (F)). Specificity is provided through display of a single-chain antibody (scFv) as targeting domain by fusion to the MV-H protein. As an alternative to scFv, designed ankyrin repeat proteins (DARPins) can be selected to become high-affinity binders to any kind of target molecule. In this study six HER2/neu-specific DARPins exhibiting different affinities and binding to different HER2/neu epitopes were applied as targeting domains. All H-DARPin fusion proteins were efficiently expressed on the cell surface. Upon coexpression with F, syncytia formation was observed in HER2/neu positive cells only and correlated directly with the HER2/neu receptor density. All H-DARPin proteins incorporated into LVs, albeit at different levels. The vectors only transduced HER2/neu-positive cells, while HER2/neu-negative cells remained untransduced. Highest titers were observed with one particular DARPin binding to the membrane distal domain of HER2/neu with medium affinity. When applied in vivo systemically, HER2/neu-targeted LVs showed exclusive gene expression in HER2/neu positive tumor tissue, while vesicular stomatitis virus-glycoprotein (VSV-G) pseudotyped vectors mainly transduced cells in spleen and liver. Thus, DARPins are a promising alternative to scFvs for retargeting of LVs.

Lentiviral vectors with measles virus glycoproteins - dream team for gene transfer?

Lentiviral vectors are potent gene transfer vehicles frequently applied in research and lately also in clinical applications. Recent improvements have come from combining lentiviral vectors with engineered envelope proteins, which now allow targeting of cell entry to any cell population of interest, as well as the transduction of quiescent cells of the haematopoietic system. We propose that measles virus envelope glycoproteins are especially well suited for this purpose because they can mediate pH-independent cell entry at the cell surface membrane and can induce cytoskeleton rearrangements that facilitate the transport of lentiviral core particles to the cell nucleus. Lentiviral vectors pseudotyped with measles virus glycoproteins are expected to improve the safety and efficacy of gene transfer to human cells.

Pseudotyping lentiviral vectors with the wild-type measles virus glycoproteins improves titer and selectivity

We pseudotyped HIV-1 vectors with cytoplasmic tail-truncated envelope glycoproteins of a wild-type (WT) measles virus (MV). The particles entered the lymphatic cells exclusively through the signaling lymphocyte activation molecule (SLAM, CD150), whereas particles pseudotyped with the MV vaccine strain glycoproteins also recognized the ubiquitous membrane cofactor protein (CD46) as receptor and had less specific cell entry. MVWT-HIV vectors reached titers of 108 t.u. ml−1, which were up to 10-fold higher than those of MVVac-HIV vectors, and discriminated between SLAM-positive and SLAM-negative cells, also in mixed cell cultures. As these vectors transduce primary human cells more efficiently than vesicular stomatitis virus-G pseudotyped vectors do, they are promising candidates for gene transfer to human lymphocytes and certain epithelial cells.

Displaying High-affinity Ligands on Adeno-associated Viral Vectors Enables Tumor Cell-specific and Safe Gene Transfer

Gene transfer vectors derived from the adeno-associated virus (AAV) have recently received increasing attention due to substantial therapeutic benefit in several clinical trials. Nevertheless, their great potential for in vivo gene therapy can only be partially exploited owing to their broad tropism. Current cell surface targeting strategies expanded vector tropism towards transduction of cell types that are inefficiently infected naturally, but failed to restrict or fully re-direct AAVs tropism. Hypothesizing that this limitation can be overcome by equipping natural receptor-blinded AAV vectors with high-affinity ligands, we displayed designed ankyrin repeat proteins (DARPin) as VP2 fusion proteins on AAV capsids ablated for natural primary receptor binding. These second generation targeting vectors demonstrated an as of yet unachieved efficiency to discriminate between target and non-target cells in mono- and mixed cultures. Moreover, DARPin-AAV vectors delivered a suicide gene precisely to tumor tissue and substantially reduced tumor growth without causing fatal liver toxicity. The latter caused death in animals treated with conventional AAV vectors with unmodified capsids, which accumulated in liver tissue and failed to affect tumor growth. This novel targeting platform will be key to translational approaches requiring restricted and cell type-specific in vivo gene delivery.

Specific Elimination of CD133+ Tumor Cells with Targeted Oncolytic Measles Virus

Tumor-initiating cells (TIC) are critical yet evasive targets for the development of more effective antitumoral strategies. The cell surface marker CD133 is frequently used to identify TICs of various tumor entities, including hepatocellular cancer and glioblastoma. Here, we describe oncolytic measles viruses (MV) retargeted to CD133. The viruses, termed MV-141.7 and MV-AC133, infected and selectively lysed CD133(+) tumor cells. Both viruses exerted strong antitumoral effects on human hepatocellular carcinoma growing subcutaneously or multifocally in the peritoneal cavity of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Notably, the CD133-targeted viruses were more effective in prolonging survival than the parental MV-NSe, which is currently assessed as oncolytic agent in clinical trials. Interestingly, target receptor overexpression or increased spreading kinetics through tumor cells were excluded as being causative for the enhanced oncolytic activity of CD133-targeted viruses. MV-141.7 was also effective in mouse models of orthotopic glioma tumor spheres and primary colon cancer. Our results indicate that CD133-targeted measles viruses selectively eliminate CD133(+) cells from tumor tissue, offering a key tool for research in tumor biology and cancer therapy.