Table 2.
Summary of the efficacy of the different intrabodies against HD
| Antibody | Target of the peptide | Model | Population | Way of administration | End point | Method of evaluation | Results |
|---|---|---|---|---|---|---|---|
| C4 intrabody | N17 terminal region | Cell culture (Lecerf et al., 2001) [44] | COS-7; BHK-21; HEK293 | Co-transfection: hHtt17aa-25/73/103Q-GFP ± C4 intrabody (ratio 5:1) | Aggregation | % of aggregate-positive transfected cells | Reduction up to 86 % |
| Organotypic cultures (Murphy and Messer, 2004) [46] | Cortico-striatal slice cultures | Malonate treatment, and transfection with hHtt17aa-25/72Q-GFP ± C4 intrabody | Cell survival | % of co-transfected died or dying cells | Rescue to wt level | ||
| Drosophila model (Wolfgang et al., 2005) [45] | ELAV-Gal4; UAS-hHttex1-20/93Q; | Genetic cross: UAS-C4 intrabody” | Survival | % of survival to adult (eclosion); mean, median, and maximal lifespan | Increased survival to adulthood (from 23 % to 100 %); increased mean adult lifespan by 30-50 % | ||
| Aggregation; quantification of soluble polyQ forms | Immunostaining; detergent-soluble hHttex1-93Q detection (Western blot) | Slowing of visible aggregate formation. Increased levels of soluble Htt | |||||
| Neurodegeneration | Photoreceptors/ommatidium quantification | Slowing of neurodegeneration in photoreceptors cells | |||||
| Mouse model (Snyder-Keller et al., 2010) [47] | B6.HD6/1 125Q (hHttex1-125Q) | C4 intrabody with AAV vector into the striatum; presymptomatic (injection: wk5 to 8 ; killed at wk16 to 32); symptomatic (injection wk 10 to 24; killed 8 to 10 wk later) | Aggregation | Immunostainig: number and size aggregates | Pre-symptomatic and symptomatic effect: aggregate reduction (size > number), more important in pre-symptomatically treated mice | ||
| VL12.3 intrabody | N17 terminal region | Cell culture (Colby et al., 2004) [48] | HEK293 | Co-tranfection: hHttex1-97Q-GFP + empty vector or VL12.3 | Aggregation | Immunostaining | 50 % reduction of aggregates vs empty vector |
| Cell culture (Southwell et al., 2008) [49] | HEK293 | Co-transfection: hHttex1-103Q-GFP + empty vector or VL12.3 | Aggregation | Immunostaining | Dose-dependent aggregate reduction | ||
| Cell survival | % of co-transfected dead cells | Reduced cell toxicity | |||||
| Co-transfection: hHttex1-25/103Q-GFP + VL12.3 | Quantification of soluble and insoluble hHttex1 | Centrifugation and Immunoblot assay | Significant reduction of insoluble but not of soluble hHttex1-103Q levels | ||||
| Co-transfection: hHttex1-103Q -SNAP tag ± VL12.3 | hHttex1-103Q turnover | Fluorescence intensity of SNAP-tag | No effect on polyQ turnover | ||||
| Cortico-striatal brain slice model (Southwell et al., 2008) [49] | Rat brain slices | Co-transfection: YFP as morphometric marker ± hHttex1-103Q -CFP ± VL12.3 | Neurodegeneration | Immunostaining: counting of healthy striatal medium spiny neurons (MSNs) | Rescue of neurodegeneration at the level of wt cells | ||
| ST14A striatal precursor cells | Co-transfection: hHttex1-103Q -GFP ± VL12.3 | hHttex1-103Q localisation and turnover | Immunostaining: cytoplasmic/nuclear hHttex1-103Q ratio | Altered cytoplasmic/nuclear trafficking: significant increase of nuclear Htt | |||
| Mouse model (Southwell et al., 2009) [51] | C57BL/6 (lentiviral HD model) | HD model: Unilateral striatal injection: hHttex1-103Q -GFP or GFP lentivirus.Treatment: + VL12.3- AAV or GFP (4 wk-old mice). Tests 6wks later. | Amphetamine-induced rotation | Ipsilateral rotations tested during 30’ after intraperitoneal amphetamine injection. | Strong reduction of the number of ipsilateral rotations to the levels of GFP lentivirus injected animals | ||
| MSNs loss | DARPP-32 staining | Rescue to the levels of GFP lentivirus injected animals | |||||
| Aggregation | Striatal immunostaining with anti-Htt MW8 (detect aggregates only) | Significant aggregate reduction vs AAV-GFP injected animals | |||||
| YAC128 (Full length-hHtt-128Q) | 2-months-old male mice and wt littermates injected bilaterally in the striatum with GFP- or VL12.3- AAV | Motor performances | Rotarod latency to fall (monthly from 3 to 7 months of age) | No effect | |||
| Beam-crossing performance (monthly from 3 to 7 months of age) | No effect | ||||||
| Climbing time (7-month-old mice) | No effect | ||||||
| Cognitive performances (spatial and cortical learning) | Novel object location and novel object preference tests (7-month-old mice) | No effect in both tests | |||||
| Anxiety | Open field test | Non significant amelioration | |||||
| Brain atrophy | Ventricular size assessment (7-month-old mice) | No effect | |||||
| Body weight | Assessment monthly from 3 to 7 months of age | No effect | |||||
| R6/2 (hhttex1- 144Q) | 3-day-old male mice and wt littermates: bilateral injection at the center of each forebrain hemisphere of GFP- or VL12.3-AAV | Motor performances | Rotarod latency to fall (weekly from w4 to death) | Reduced latency to fall (wk 10 to 12) | |||
| Beam-crossing performance (weekly from w4 to death) | No rescue: Increased severity of the phenotype (time to cross the beam) | ||||||
| Brain atrophy | Ventricular size assessment (10-wk-old mice) | No effect | |||||
| Body weight | Assessment weekly from 4 wk until death | No effect | |||||
| Aggregation | Striatal immunostaining with anti-Htt MW8 (detect aggregates only) and nuclear marker; counting of positive foci (10 week-old mice) | Reduction of the number of neuropil aggregates; no significant reduction of intranuclear aggregates | |||||
| Life span | Once ill, twice a day assessment of righting reflex | Aggravation and decrease survival | |||||
| MW7 intrabody | Poly P region | Cell culture (Khoshnan et al., 2002) [50] | HEK293 | Co-transfection: hHttex1-97Q-GFP and MW7 or empty vector | Aggregated/soluble Htt | Centrifugation, SDS treatment and western blotting | Reduction of both aggregated and soluble polyQHtt |
| Cell survival | TUNEL staining | 33 % reduction of TUNEL positive cells | |||||
| Cell culture (Southwell et al., 2008) [49] | HEK293 | Co-transfection: hHttex1-103Q-GFP + empty vector or MW7 | Aggregation | Immunostaining | Aggregate reduction with a threshold-effect | ||
| Cell survival | % of co-transfected dead cells | Reduced cell toxicity | |||||
| Co-transfection: hHttex1-25/103Q-GFP + MW7 | Quantification of soluble and insoluble hHttex1 | Centrifugation and Immunoblot essay | Significant reduction of both soluble and insoluble hHttex1-103Q; no effect on soluble wt hHttex1-25Q | ||||
| Co-transfection: hHttex1-103Q-SNAP tag ± MW7 | hHttex1-103Q turnover | Fluorescence intensity of SNAP tag | Significant decreased fluorescence (increased hHttex1-103Q turnover) | ||||
| Cortico-striatal brain slice model (Southwell et al., 2008) [49] | Rat brain slices | Co-transfection: YFP ± hHttex1-103Q -CFP ± MW7 | Neurodegeneration | Immunostaining: counting of healthy MSNs | Non-significant reduction of neurodegeneration | ||
| ST14A striatal precursor | Co-transfection: hHttex1-103Q -GFP ± MW7 | hHttex1-103Q localisation and turnover | Immunostaining: cytoplasmic/nuclear hHttex1-103Q ratio | No effect | |||
| Happ1-Happ3 antibodies | Poly P region | Cell culture (Southwell 2008) [49] | HEK293 | Co-transfection: hHttex1-103Q-GFP + empty vector or Happ1-Happ3 | Aggregation | Immunostaining | Dose-dependent aggregate reduction |
| Cell survival | % of co-transfected dead cells | Reduced cell toxicity | |||||
| Co-transfection: hHttex1-25/103Q-GFP + Happ1-Happ3 | Quantification of soluble and insoluble hHttex1 | Centrifugation and Immunoblot essay | Significant reduction of both soluble and insoluble hHttex1-103Q; no effect on soluble wt hHttex1-25Q | ||||
| Co-transfection: hHttex1-103Q-SNAP tag ± Happ1-Happ3 | hHttex1-103Q turnover | Fluorescence intensity of SNAP tag | Significant decreased fluorescence (increased hHttex1-103Q turnover) | ||||
| Cortico-striatal brain slice model (Southwell et al., 2008) [49] | Rat brain slices | Co-transfection: YFP as morphometric marker ± hHttex1-103Q -CFP ± Happ1-Happ3 | Neurodegeneration | Immunostaining: counting of MSNs | Significant reduction of neurodegeneration | ||
| ST14A striatal precursor | Co-transfection: hHttex1-103Q -GFP ± Happ1-Happ3 | hHttex1-103Q localisation and turnover | Immunostaining: cytoplasmic/nuclear hHttex1-103Q ratio | No effect | |||
| Mouse model (Southwell et al., 2009) [51] | C57BL/6 (lentiviral HD model) | HD model: Unilateral striatal injection: hHttex1-103Q -GFP or GFP lentivirus.Treatment: + GFP- or Happ1- AAV (4 wk-old mice). Tests 6wks later. | Amphetamine-induced rotation, | Ipsilateral rotations tested during 30’ after intraperitoneal amphetamine injection. | Strong reduction of the number of ipsilateral rotations to the levels of GFP lentivirus injected animals | ||
| MSNs loss | DARPP-32 staining | Rescue to the levels of GFP lentivirus injected animals | |||||
| Aggregation | Striatal immunostaining with anti-Htt MW8 (detect aggregates only) and nuclear marker; counting of positive foci. | Significant aggregate reduction vs AAV-GFP injected animals | |||||
| YAC128 (Full length-hHtt-128Q) | 2-months-old male mice and wt littermates injected bilaterally in the striatum with GFP- or Happ1- AAV | Motor performances | Rotarod latency to fall (monthly from 3 to 7 months of age) | Improvement in 3-, 4-, and 7 –month-old mice | |||
| Beam-crossing performance (monthly from 3 to 7 months of age) | Partial improvement | ||||||
| Climbing (7-month-old mice) | Increased climbing time to the level of wt littermates | ||||||
| Cognitive performances (spatial and cortical learning) | Novel object location and novel object preference tests (7-month-old mice) | Significant amelioration of spatial and cortical learning | |||||
| Anxiety | Open field test | Rescue to the level of wt littermates | |||||
| Brain atrophy | Ventricular size assessment (7-month-old mice) | Reduction of ventricular size | |||||
| Body weight | Assessment monthly from 3 to 7 months of age | No effect | |||||
| R6/2 (hhttex1- 144Q) | 3-day-old male mice and wt littermates: bilateral injection at the center of each forebrain hemisphere of GFP- or Happ-AAV | Motor performances | Rotarod latency to fall (weekly from 4 wk until death) | Amelioration (between w9 and 12 of age) vs GFP-AVV injected animals. | |||
| Beam-crossing performance (weekly from 4 wk until death) | Reduction of the time to cross the 12 mm beam in 10- and 11-week-old mice, and the 6 mm beam between 9 and 11 weeks of age | ||||||
| Brain atrophy | Ventricular size assessment (10-wk-old mice) | Reduction of ventricular size to the level of wt littermates | |||||
| Body weight | Assessment weekly from 4 wk until death | No effect | |||||
| Aggregation | Striatal immunostaining with anti-Htt MW8 (detect aggregates only) and nuclear marker; counting of positive foci (10 week-old mice). | Reduction of the number of both neuropil and intranuclear aggregates. | |||||
| Life span | Once ill, twice a day assessment of righting reflex | No effect | |||||
| N171-82Q: hHtt171aa-82Q | Four-week old male mice and wt littermates: bilateral striatal injection of GFP- or Happ1-AAV | Motor performances | Latency to fall with accelerating rotarod (every 2 wks from wk6 until death) | Significant improvement from wk 20 to 40 at the level of wt mice | |||
| Beam-crossing performance (every 2 wks from wk6 until death) | Significant improvement with reduction of time to cross the three beams vs GFP-AAV treated and wt mice. | ||||||
| Clasping (22-week-old mice) | Attenuation of clasping behavior | ||||||
| Body weight | Assessment every 2 wks from wk6 until death | Increased weight vs GFP-AAV treated but not to the level of wt littermates (from wk 22) | |||||
| Life span | Once ill, twice a day assessment of righting reflex | 33 % increase of maximum life-span (from 30 to 40 wk) vs GFP-AAV treated mice. | |||||
| BACHD: Full-length- hHtt-97Q- | 2-months-old male mice and wt littermates: bilateral striatal injection of GFP- or Happ1-AAV | Motor performances | Rotarod latency to fall (monthly from month 3 to 6) | Increased latency to fall in 5- and 6- month-old mice | |||
| Beam-crossing performances (monthly from month 3 to 6) | Decrease time to cross the beams at 5 and 6 months (28 mm beam) and at month 6 (6 mm beam) | ||||||
| Climbing time (6-month-old mice) | Increased climbing time vs GFP treatment | ||||||
| Cognitive performances (spatial and cortical learning) | Novel object location and novel object preference tests (6-month-old mice) | No effect | |||||
| Anxiety | Open field test | Significant effect vs GFP-AAV treated mice | |||||
| Brain atrophy | Ventricular size assessment (6-month-old mice) | Reduction of ventricular size | |||||
| Body weight | Assessment monthly (from 3 to 6 months of age) | No effect | |||||
| mEM48 intrabody (Wang et al., 2008) [52] | VA residues after the polyP region | Cell culture | HEK293 | Co-transfection: hHtt208aa 23/130Q ± EM48 | Cell survival | % of co-transfected dead cells | Improved cell viability |
| Rat cortical neurons | Co-transfection: hHtt208aa 23/130Q ± EM48 | Neuritic disruption and pyknotic nuclei | Neuronal morphology | Significant reduction of transfected neurons with disrupted neurites or fragmented nuclei | |||
| PC12 cells | Transfection of hHtt208aa 23/130Q ± AAV-EM48 | Neuropil aggregates | Immunostaining | Significant reduction of neuropil aggregates | |||
| Mouse model | R6/2 (hhttex1- 144Q) | Intrastriatal injection of helper dependent AAV EM48 (7-wk-old mice) | Neuropil aggregates | Immunostaining (4 wk after injection) | Significant less neuropil aggregates vs non injected region; no effect on intranuclear inclusion | ||
| N171-82Q | Bilateral striatal injection of helper dependent AAV EM48 (10-wk-old mice) | Neuropil aggregates | Immunostaining (6 wk after injection) | Significant less neuropil aggregates vs non injected region; no effect on intranuclear inclusions | |||
| Motor performances | Stride length (8-wk post injection) | Improvement | |||||
| Rotarod latency to fall (8-wk post injection) | Significant improvement | ||||||
| Body weight | No effect | ||||||
| Survival | No effect | ||||||
| Monoclonal antibodies 1C2 (Heiser et al., 2000 [43]; Trottier et al., 1995) [42] | PolyQ chain (soluble) | Cell culture | COS-1 | Co-transfection: hHttex1-51Q ± 1C2 | Aggregation | Filter retardation assay | Up to 85 % reduction in aggregates |
Legend: To characterize Htt fragments we use the general indication HttXaa-YQ: the length of the fragment is expressed as a number X of amino acids (aa) (superimposed); the length of polyQ expansion is expressed as a number Y of Q.